Dried compositions containing flap endonuclease

ABSTRACT

There is disclosed a composition of an aqueous solution comprising, consisting or consisting essentially of a flap endonuclease, a bulking agent and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less and wherein the composition is substantially free of glycerol.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of each of provisionals62/508,975, filed May 19, 2017, 62/508,990, filed May 19, 2017,62/540,478 filed Aug. 2, 2017, each incorporated by reference in itsentirety for all purposes.

BACKGROUND

Commercial kits for performing nucleic acid based assays often containreagents such as enzymes, nucleotides, detergents, buffers, primers,probes and inorganic salt, including MnCl₂, MgCl₂, NaCl, and KCl (Inniset al, (1990) PCR Protocols: A Guide to Methods and Applications, Ch. 1,Optimizations of PCRs).

Nucleic-acid-based assays can be “nucleic acid amplification-basedassays,” i.e., assays that utilize one or more steps for amplifying anucleic acid target sequence. Various amplification methods for use innucleic acid based assays are known in the art. Alternatively, anucleic-acid-based assay can be a “non-nucleic acid amplification-basedassay,” i.e., an assay that does not rely on any step for amplifying anucleic acid target sequence. An exemplary non-nucleic acidamplification-based assay is a “cleavage-based assay,” which is an assaythat relies on specific cleavage, by a flap endonuclease, of a linearduplex cleavage structure formed by the specific hybridization ofoverlapping oligonucleotides to a target nucleic acid. In these assays,an invader oligonucleotide (also referred to as an “invader probe”) isdesigned to stably anneal to a target nucleic acid at assay reactiontemperature. Signal probe oligonucleotides containing a targethybridizing sequence and a non-target-hybridizing flap region thatoverlays the invader oligonucleotide, are designed with a melttemperature that is approximately that of the assay temperature. As aconsequence, the signal probes are in a state of annealing/dissociatingfrom the target nucleic acid at reaction conditions. When a signal probeis annealed to the target nucleic acid in the presence of a flapendonuclease, the non-target hybridizing flap region is cleaved in anoverlap-dependent manner by the flap endonuclease to release a cleavageproduct. The cleaved flap region then anneals with a hairpin configuredprobe comprising a signalling moiety and a quenching moiety (oftenreferred to as a “FRET probe”) to form an overlap between a portion ofthe cleaved flap portion and the portion of the FRET probe that isjoined to one of the signalling moiety or the quenching moiety. In thepresence of a flap endonuclease, the FRET probe is cleaved in anoverlap-dependent manner to release one of the signalling moiety orquenching moiety from the FRET probe, thereby resulting in a detectablesignal. Because an excess of signal probe and FRET probe relative to theinvader probe is used in these assays, they are often referred to assignal amplification assays. The principles of cleavage-based assays arewell-known in the art, and exemplary assays are described in, forexample, Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan etal. (Mol. Diagn. 4: 135-144, 1999), Allawi et al. (J. Clin. Microbiol.44:3443-3447, 2006), U.S. Pat. Nos. 5,846,717 & 6,706,471 to Brow etal., and U.S. Pat. No. 5,614,402 to Dahlberg et al. Cleavage-basedassays include, e.g., the commercially available Invader® assays(Hologic, Inc., Marlborough, Mass.).

In some circumstances, it can be desirable to carry out“amplification-based assays” (e.g. PCR) and “non-amplification-basedassays” (e.g. assays using Cleavase® enzyme) in the same reaction.Cleavase® enzyme is a thermostable structure-specific endonuclease thatcleaves at the junctions between single- and double-strandeddeoxyribonucleic.

Magnesium ions have been reported to increase activity of polymerasesand other enzymes. Potassium chloride has been reported to facilitatenucleic acid hybridizations. A number of inorganic salts have beenreported to protect proteins under various conditions of stressincluding heat, chaotropic agent exposure and lyophilization (Liu et al(2007) FEBS Letters. 581:1047; Kanaya et al (1996) J. Biol. Chem.271:32729; Innis et al, (1990) PCR Protocols: A Guide to Methods andApplications, Ch. 1, Optimizations of PCRs; Menendez et al (1998) J.Biol. Chem. 273:167; Janeway et al (1993) Biochemistry. 32:1601, Fox etal (1971) J. Biol. Chem. 246:5739, Chang et al (2002) J. Biol. Chem.277:277:4663, Rutter et al (1958) J. Biol. Chem. 233:374, Huszar et al(1981) J. Virol. 37:580-588, Wang (2000) Int. J. Pharmaceutics.203:1-60). Salts are also used in “amplification-based assays” and“non-amplification-based assays” assays. By way of example, typicalreaction conditions to carry out the Invader® assay include potassiumchloride in the enzyme stock and magnesium chloride in the reactionbuffer.

However, the stability of a lyophilized substance is affected by thehygroscopicity of any salts present in the lyophilized cake.Hygroscopicity of a lyophilized substance in turn affects the timeavailable to package the lyophilized substance, and affects the durationand conditions under which the lyophilized substance can be stored andshipped. The undesired rehydration of a lyophilized substance negativelyimpacts the activity of lyophilized components. To minimize the negativeimpact from undesired rehydration of a lyophilized substance, long termstorage of such substances is usually performed with refrigeration.

The present invention seeks to provide improvements in lyophilizedcompositions, especially when used in nucleic acid based assays—such asnon-amplification-based assays or non-amplification-based assayscombined with amplification based assays.

SUMMARY

There is disclosed a composition of an aqueous solution comprising,consisting or consisting essentially of a flap endonuclease, a bulkingagent and an organic buffer, wherein the aqueous solution has aninorganic salt concentration of 6 mM or less and wherein the compositionis substantially free of glycerol.

Suitably, the aqueous solution further comprises at least oneoligonucleotide useful for performing a molecular assay.

Suitably, the aqueous solution further comprises at least oneoligonucleotide useful for performing a nucleic acid based assay.

Suitably, the flap endonuclease is a Cleavase® enzyme.

Suitably, the composition further comprises at least one polymerase.

Suitably, the at least one polymerase includes a polymerase present inthe aqueous solution at a concentration from about 0.10 U/ul to about0.25 U/ul in the aqueous solution.

Suitably, the at least one polymerase includes a polymerase present inthe aqueous solution at a concentration selected from: 0.11 U/ul, 0.12U/ul, 0.14 U/ul, 0.146 U/ul, 0.1687 U/ul, 0.2 U/ul, and 0.022 U/ul.

Suitably, the at least one polymerase includes a polymerase that is ahot-start polymerase.

Suitably, the hot-start polymerase is a recombinant Taq DNA polymerasebound by an antibody that specifically blocks polymerase activity of thepolymerase.

Suitably, the hot-start polymerase is a chemically modified recombinantTaq DNA polymerase, wherein the chemical modification inhibitspolymerase activity of the polymerase.

Suitably, the at least one polymerase includes a reverse transcriptasepresent in the aqueous solution, suitably, at a concentration from about0.1 U/ul to about 4.0 U/ul.

Suitably, the reverse transcriptase is an AMV reverse transcriptase.

Suitably, the reverse transcriptase is an MMLV reverse transcriptase.

Suitably, the at least one oligonucleotide includes an invader probe.

Suitably, the sequence of the invader probe is partially or completelycomplementary to a target nucleic acid sequence.

Suitably, the at least one oligonucleotide includes a signalling probe.

Suitably, the sequence of the signalling probe is partiallycomplementary to a target nucleic acid sequence.

Suitably, the sequence of the signalling probe comprises a flap region.

Suitably, the flap region at least partially overlaps with an invaderprobe.

Suitably, the at least one oligonucleotide includes a FRET probe.

Suitably, the sequence of the FRET probe is partially complementary tothe flap region of a signalling probe.

Suitably, the FRET probe comprises a label covalently joined thereto.

Suitably, the label is a fluorescent molecule.

Suitably, the label is located at the 5′ end of the FRET probe.

Suitably, the FRET probe comprises a quencher molecule covalently joinedthereto, within quenching proximity to the fluorescent molecule, andcapable of quenching at least partially fluorescence form thefluorescent molecule.

Suitably, the at least one oligonucleotide includes a target captureprobe.

Suitably, the target capture probe has a target hybridizing portion thatspecifically or non-specifically hybridizes to a target nucleic acidunder stringent conditions.

Suitably, the target capture probe has a target hybridizing portion thatnon-specifically hybridizes to a target nucleic acid under stringentconditions.

Suitably, the non-specific target hybridizing portion of the targetcapture probe comprises randomly arranged K nucleotides or randomlyarranged R nucleotides (IUPAB-IUB ambiguity codes).

Suitably, the aqueous solution comprises two or more oligonucleotidesfor performing a multiplex molecular assay.

Suitably, the bulking agent is trehalose.

Suitably, the bulking agent is present at a concentration from about 0.2M to about 0.5 M, suitably about 0.36 M.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 6 mM or less. Suitably, the aqueous solution comprises an inorganicsalt concentration between about 6 mM to about 0.5 mM.

Suitably, the inorganic salts are present at a mass per microliter offrom about 0.373 μg/μl about 0.029 μg/μl.

Suitably, the inorganic salt is sodium chloride, suitably, wherein thesodium chloride is present at a mass per microliter of from about 0.35ug/ul to about 0.029 ug/ul, suitably about 0.32 ug/ul.

Suitably, the inorganic salt is potassium chloride, suitably, whereinthe potassium chloride is present at a mass per microliter of from about0.373 ug/ul of potassium chloride to about 0.019 ug/ul of potassiumchloride, suitably about 0.03 ug/ul.

Suitably, the aqueous solution contains from about 0.135 ug/ul of sodiumions to about 0.006 μg/μl of sodium ions, suitably, about 0.127 ug/ul.

Suitably, the aqueous solution contains from about 0.196 ug/ul ofpotassium ions to about 0.010 ug/ul of potassium ions, suitably about0.016 ug/ul.

Suitably, the aqueous solution contains from about 0.355 ug/ul chlorideions to about 0.009 ug/ul chloride ions, suitably about 0.337 ug/ul.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 4 mM or less.

Suitably, the aqueous solution comprises a mass per microliter ofinorganic salt from about 0.298 μg/μl to about 0.234 μg/μl.

Suitably, the aqueous solution comprises a mass per microliter ofchloride ions from about 0.284 μg/μl to about 0.071 μg/μl.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 3 mM or less.

Suitably, the aqueous solution comprises a mass per microliter ofinorganic salt from about 0.224 μg/μl to about 0.175 μg/μl.

Suitably, the aqueous solution comprises a mass per microliter ofchloride ions from about 0.213 μg/μl to about 0.053 μg/μl.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 2 mM or less.

Suitably, the aqueous solution comprises a mass per microliter ofinorganic salt from about 0.149 μg/μl to about 0.117 μg/μl.

Suitably, the aqueous solution comprises a mass per microliter ofchloride ions from about 0.142 μg/μl to about 0.036 μg/μl.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 1 mM or less.

Suitably, the aqueous solution comprises a mass per microliter ofinorganic salt from about 0.075 μg/μl to about 0.058 μg/μl.

Suitably, the aqueous solution comprises a mass per microliter ofchloride ions from about 0.071 μg/μl to about 0.018 μg/μl.

Suitably, the aqueous solution comprises an inorganic salt concentrationof 500 μM or less.

Suitably, the aqueous solution comprises a mass per microliter ofinorganic salt from about 0.037 μg/μl to about 0.029 μg/μl.

Suitably, the aqueous solution comprises a mass per microliter ofchloride ions from about 0.036 μg/μl to about 0.009 μg/μl.

Suitably, the inorganic salt concentration of the aqueous solution isless than 1 mM sodium chloride.

Suitably, the aqueous solution does not contain sodium chloride.

Suitably, the aqueous solution contains less than 1 mM magnesium ions.

Suitably, the aqueous solution contains less than 0.1 mM magnesium ions.

Suitably, the aqueous solution further comprises deoxynucleotidetriphosphates (dNTPs).

Suitably, the dNTPs include dATP at a concentration of from 0.1 mM to0.5 mM in the aqueous solution, suitably about 0.28 mM to about 0.46 mM.

Suitably, the dATP is at a concentration of 0.3 mM to 0.4 mM in theaqueous solution, e.g, 0.375 mM.

Suitably, the dNTPs include dGTP at a concentration of from 0.1 mM to0.4 mM in the aqueous solution.

Suitably, the dGTP is at a concentration of 0.3 mM to 0.4 mM in theaqueous solution, suitably 0.29 to 0.46 mM, e.g, 0.375 mM.

Suitably, the dNTPs include dCTP at a concentration of from 0.1 mM to0.4 mM in the aqueous solution, suitably 0.29 to 0.46 mM.

Suitably, the dCTP is at a concentration of 0.3 mM to 0.4 mM in theaqueous solution, e.g., 0.375 mM.

Suitably, the dNTPs include dTTP at a concentration of from 0.1 mM to0.4 mM in the aqueous solution, suitably 0.2 to 0.37 mM, e.g, 0.284 mM.

Suitably, the dNTPs include dUTP at a concentration of from 0.1 mM to0.4 mM in the aqueous solution, suitably 0.125 to 0.234 mM, e.g., 0.182mM.

Suitably, the flap endonuclease is present in the aqueous solution atabout 0.010 μg/μl to about 0.050 ug/ul, suitably at about 0.12 ug/ul to0.047 ug/ul.

Suitably, the flap endonuclease is present in the aqueous solution atabout 0.030 μg/μl to about 0.04 μg/μl, suitably, wherein the flapendonuclease is present in the aqueous solution at about 0.030 μg/μl toabout 0.035 μg/μl.

Suitably, the organic buffer is 3-(N-morpholino)propanesulfonic acid(MOPS) buffer.

Suitably, the MOPS buffer is present at a concentration of from 10 to 20mM in the aqueous solution, suitably at a concentration of 12.5 mM to 20mM in the aqueous solution.

Suitably, the organic buffer is tris(hydroxymethyl)aminomethane (Tris)buffer.

Suitably, the Tris buffer is present at a concentration of from 40 mM to60 mM in the aqueous solution, suitably at a concentration of 50 mM inthe aqueous solution.

Suitably, the composition contains a globular protein.

Suitably, the globular protein is bovine serum albumin (BSA).

Suitably, the bovine serum albumin (BSA) is non-acetylated BSA,suitably, ultrapure non-acetylated BSA.

Suitably, globular protein is present in an amount of 0.40 to 0.60μg/μl, suitably, 0.50 μg/μl.

Suitably, the composition comprises, consists or consists essentially ofa Cleavase® enzyme, trehalose, MOPS buffer, dNTPs, an inorganic saltconcentration of 5 mM or less and wherein the composition issubstantially free of glycerol.

Suitably, the composition comprises, consists or consists essentially ofa Cleavase® enzyme present in the aqueous solution at about 0.030 μg/μl,trehalose present in a concentration of about 0.3M, MOPS buffer in aconcentration of about 12.5 mM, dNTPs at a concentration of about 0.3 mMeach, optionally, dATP, dGTP, and dCTP are at about 0.375 mM, dTTP isabout 0.284 mM and dUTP is about 0.182 mM, an inorganic saltconcentration of 5 mM or less and wherein the composition issubstantially free of glycerol.

Suitably, the composition comprises, consists or consists essentially ofa Cleavase® enzyme, trehalose, Tris buffer, dNTPs, bovine serum albumin,an inorganic salt concentration of 5 mM or less and wherein thecomposition is substantially free of glycerol.

Suitably, the composition comprises, consists or consists essentially ofa Cleavase® enzyme present in the aqueous solution at about 0.030 μg/μl,trehalose present in a concentration of about 0.3M, Tris buffer in aconcentration of about 50 mM, dNTPs at a concentration of about 0.3 mMeach, optionally, dATP, dGTP, and dCTP are at about 0.375 mM, dTTP isabout 0.284 mM and dUTP is about 0.182 mM, bovine serum albumin at about0.5 μg/μl, an inorganic salt concentration of 5 mM or less and whereinthe composition is substantially free of glycerol.

Suitably, the composition comprises, consists or consists essentially ofCleavase®, trehalose, MOPS buffer, dNTPs, bovine serum albumin, aninorganic salt concentration of 5 mM or less and wherein the compositionis substantially free of glycerol.

Suitably, the composition comprises, consists or consists essentially ofCleavase® present in the aqueous solution at about 0.035 μg/μl,trehalose present in a concentration of about 0.36M, MOPS buffer at aconcentration of about 15 mM, dNTPs at a concentration of about 0.38 mMeach, bovine serum albumin at about 0.5 μg/μl, an inorganic saltconcentration of 5 mM or less and wherein the composition issubstantially free of glycerol.

Suitably, the composition includes alpha-cyclodextrine, for example at aconcentration of 0.1-0.5 ug/ml.

There is also disclosed a dried form of the composition according to thepresent disclosure. By way of example, 24 ul of a an aqueous solutiondescribed herein is dried to provide a dried composition having a massin the range of from about 0.003 g to about 0.004 g, from about 0.0032 gto about 0.0037 g, 0.0033 g, 0.0034 g, 0.0035 g, or 0.0036 g. It isunderstood that a plurality of dried compositions made from aliquots ofa single bulk aqueous solution will have variations in the mass of eachdried composition. By way of example, two or more 24 ul aliquots of abulk aqueous solution will each be dried to separately provide a driedcomposition having a mass in the range of from about 0.003 g to about0.004 g. Thus, the average weight of each of the plurality of driedpellets is in the range of from about 0.003 g to about 0.004 g, fromabout 0.0032 g to about 0.0037 g, 0.0033 g, 0.0034 g, 0.0035 g, or0.0036 g. Changing the concentration of a component in the aqueoussolution will result in a change to the mass of the dried composition.For example, changing the concentration of an enzyme to optimize theenzyme activity for use in a particular reaction may result in a changeto the mass of the dried composition. Similarly, changes in the type ofcomponent used can result in changes in the mass of the driedcomposition. For example using different flap endonucleases can requireconcentration changes to provide a desired enzyme activity level.Changing the concentration and/or type of one or more of the variouscomponents is understood to fall within the instant disclosure, and thusresultant changes to the mass of the dried composition is similarlyunderstood to fall within the instant disclosure.

There is also disclosed a dried composition comprising, consisting orconsisting essentially of a flap endonuclease, a bulking agent and anorganic buffer, wherein the one or more inorganic salts are present inthe dried composition at a mass that is 0.350% or less of the total massof the dried composition and wherein the dried composition issubstantially free of glycerol.

Dried compositions are useful for nucleic acid based assays followingreconstitution. Dried compositions surprisingly provide robust resultsfollowing prolonged exposure to humid environments. The dried form ofthe compositions are useful in nucleic acid based assays followingexposure to a humid environment, wherein the absolute humidity level ofthe humid environment is greater than 2.3 grams of water per cubic meterof air for a period of time of up to 3 hours; preferably for a period oftime from 90 minutes to 180 minutes; preferably about 90 minutes; ofpreferably about 180 minutes. Dried compositions surprisingly providerobust results following prolonged incubation times of the pre-driedaqueous solution.

Suitably, one or more inorganic salts are present in the driedcomposition at a mass that is in from about 0.311% to about 0.024% ofthe total mass of the dried composition.

Suitably, the one or more inorganic salts are selected from the groupconsisting of: sodium chloride, potassium chloride and both sodiumchloride and potassium chloride.

Suitably, the dried composition further comprises at least oneoligonucleotide useful for performing a molecular assay.

Suitably, the dried composition further comprises at least oneoligonucleotide useful for performing a nucleic acid based assay.

Suitably, the at least one oligonucleotide includes a probeoligonucleotide, suitably at least two probe oligonucleotides.

Suitably, the probe oligonucleotide(s) is partially or completelycomplementary to a target nucleic acid sequence.

Suitably, the flap endonuclease is a Cleavase® enzyme.

Suitably, the dried composition further comprises at least two probeoligonucleotides capable of annealing to a target nucleic acid to form athree-dimensional structure that can be recognized by the flapendonuclease.

Suitably, the dried composition further comprises at least onepolymerase.

Suitably, the at least one polymerase includes a polymerase present inthe aqueous solution at a concentration from about 0.10 U/μl to about0.2 U/μl in the aqueous solution.

Suitably, the at least one polymerase includes a polymerase present inthe aqueous solution at a concentration selected from about 0.1 U/ul toabout 0.25 U/ul.

Suitably, the at least one polymerase includes a polymerase that is ahot-start polymerase.

Suitably, the hot-start polymerase is a recombinant Taq DNA polymerasebound by an antibody that specifically blocks polymerase activity of thepolymerase.

Suitably, the hot-start polymerase is a chemically modified recombinantTaq DNA polymerase, wherein the chemical modification inhibitspolymerase activity of the polymerase.

Suitably, the at least one polymerase includes a reverse transcriptasepresent in the aqueous solution, suitably, at a concentration from about0.1 U/μl to about 0.6 U/μl.

Suitably, the reverse transcriptase is an AMV reverse transcriptase.

Suitably, the reverse transcriptase is an MMLV reverse transcriptase.

Suitably, the at least one oligonucleotide includes a detection probe.

Suitably, the at least one oligonucleotide includes a detection probe.

Suitably, the sequence of the detection probe is partially or completelycomplementary to a target nucleic acid sequence.

Suitably, the detection probe comprises a label covalently joinedthereto.

Suitably, the label is a fluorescent or chemiluminescent molecule.

Suitably, the label is located at the 5′ end of the detection probe andan internal quencher molecule.

Suitably, the at least one oligonucleotide includes an invader probe.

Suitably, the sequence of the invader probe is partially or completelycomplementary to a target nucleic acid sequence.

Suitably, the at least one oligonucleotide includes a signalling probe.

Suitably, the sequence of the signalling probe is partiallycomplementary to a target nucleic acid sequence.

Suitably, the sequence of the signalling probe comprises a flap region.

Suitably, the flap region at least partially overlaps with an invaderprobe.

Suitably, the at least one oligonucleotide includes an FRET probe.

Suitably, the sequence of the FRET probe is partially complementary tothe flap region of a signalling probe.

Suitably, the FRET probe comprises a label covalently joined thereto.

Suitably, the label is a fluorescent molecule.

Suitably, the label is located at the 5′ end of the FRET probe.Suitably, the FRET probe comprises a quencher molecule covalently joinedthereto, within quenching proximity to the fluorescent molecule, andcapable of quenching at least partially fluorescence form thefluorescent molecule.

Suitably, the at least one oligonucleotide includes a target captureprobe.

Suitably, the target capture probe has a target hybridizing portion thatspecifically or non-specifically hybridizes to a target nucleic acidunder stringent conditions.

Suitably, the target capture probe has a target hybridizing portion thatnon-specifically hybridizes to a target nucleic acid under stringentconditions.

Suitably, the non-specific target hybridizing portion of the targetcapture probe comprises randomly arranged K nucleotides or randomlyarranged R nucleotides.

Suitably, the composition comprises oligonucleotides for performing amultiplex molecular assay.

Suitably, the bulking agent is trehalose.

Suitably, the dried composition further comprises deoxynucleotidetriphosphates (dNTPs).

Suitably, the organic buffer is 3-(N-morpholino)propanesulfonic acid(MOPS) buffer.

Suitably, the organic buffer is tris(hydroxymethyl)aminomethane (Tris)buffer.

Suitably, the dried composition contains a globular protein.

Suitably, the globular protein is bovine serum albumin (BSA).

Suitably, the bovine serum albumin (BSA) is non-acetylated BSA,suitably, ultrapure non-acetylated BSA.

There is also disclosed a method of forming a mixture for use inperforming a nucleic acid based assay, the method comprising combining areconstitution solution and the dried composition described herein,wherein the reconstitution solution comprises at least one inorganicsalt.

Suitably, the reconstitution solution comprises an inorganic saltconcentration of less than 1 mM.

Suitably, the reconstitution solution comprises magnesium ions.

Suitably, the reconstitution solution comprises MgCl₂ at a concentrationfrom about 5 mM to about 15 mM, suitably, from 9 mM to 10 mM or 9-12 mM,optionally 11.25 mM.

Suitably, the reconstitution solution is selected from the groupconsisting of: methyl paraben at a concentration from about 0.012% w/vto about 0.020% w/v, propyl paraben at a concentration from about 0.006%w/v to about 0.010% w/v, absolute ethanol at a concentration from about0.20% v/v to about 0.30% v/v, or a combination thereof.

Suitably, the concentration of the methyl paraben in the reconstitutionsolution is 0.016% w/v.

Suitably, the concentration of the propyl paraben in the reconstitutionsolution is 0.008% w/v.

Suitably, the concentration of the absolute ethanol is present in thereconstitution solution at about 0.26% v/v.

There is also disclosed a method for preparing a dried composition foruse in performing a nucleic acid based assay, the method comprising thesteps of: (i) freezing an aqueous solution of the present disclosure,thereby forming a frozen form of the aqueous solution; and (ii) exposingthe frozen form from step (i) to lyophilization conditions, therebyforming a dried composition.

Suitably, the dried composition is exposed to a humid environment,wherein the absolute humidity level of the humid environment is greaterthan 2.3 grams of water per cubic meter of air at 30 degrees C.

Suitably, the dried composition is exposed to the humid environment forup to 3 hours.

Suitably, the method comprises the step of storing the dried compositionin a sealed vessel.

There is also disclosed a method for preparing a dried composition foruse in performing a nucleic acid based assay, comprising the step of:drying the aqueous solution described herein using a drying methodselected from the group consisting of: dehydration, desiccation,lyophilization, and spray-drying, thereby forming a dried composition.

Suitably, the drying method is lyophilization and the dried compositionis a lyophilized composition.

Suitably, the method further comprises the step of storing the driedcomposition in a sealed vessel.

There is also disclosed a kit for use in performing a nucleic acid basedassay, the kit comprising a first vessel containing the driedcomposition as described herein, and a second vessel containing areconstitution solution comprising MgCl₂ at a concentration from about3.8 mM to about 14.4 mM, suitably, about 9.4 mM or about 11.25.

Suitably, the first vessel is a multiwell plate comprising one or morewells.

Suitably, each of the one or more wells contains a dried single unitdose pellet that contains a percent mass of inorganic salt to mass ofpellet of 0.311% or less.

Suitably, the first and second vessels are incorporated within a deviceadapted for automated transfer of the reconstitution solution from thesecond vessel into the first vessel.

Suitably, each of the one or more wells contains a dried single unitdose pellet with a weight of from about 0.000125 g to about 0.000667 gfor each microliter of aqueous solution that was dried to form the driedsingle unit dose pellet in each well.

Suitably, each of the one or more wells contains a dried single unitdose pellet each having a weight in the range of from about 0.003 g toabout 0.004 g, from about 0.0032 g to about 0.0037 g, 0.0033 g, 0.0034g, 0.0035 g, or 0.0036 g.

There is also disclosed a dialysis composition comprising, consisting orconsisting essentially of an aqueous solution containing an organicbuffer, a bulking agent, chloride ions and a chelating agent.

Suitably, the bulking agent is trehalose.

Suitably, the bulking agent is present at a concentration from about 100mM to 300 mM, suitably, 200 mM.

Suitably, the organic buffer is tris(hydroxymethyl)aminomethane (Tris)buffer.

Suitably, the Tris buffer is present at a concentration of from 10 mM to30 mM in the aqueous solution, suitably at a concentration of 20 mM inthe aqueous solution. Suitably, the pH is about 8.0.

Suitably, the chloride ions are KCl.

Suitably, the KCl is present at a concentration of about 40 to 60 mM,suitably, 50 mM.

Suitably, the chelating agent is EDTA.

Suitably, the EDTA is present in the aqueous solution at a concentrationfrom 0.05 to 0.2 mM, suitably, 0.1 mM.

Suitably, the composition comprises a flap endonuclease, suitably, aCleavase® enzyme.

There is also disclosed a method for preparing a substantiallyglycerol-free flap endonuclease composition comprising: (i) providing anaqueous solution comprising, consisting or consisting essentially of aflap endonuclease and glycerol; (ii) dialysing the aqueous solution intothe dialysis composition as described herein; and (iii) obtaining asubstantially glycerol-free flap endonuclease composition.

Suitably, the aqueous solution in step (i) contains from about 0.0 toabout 0.5% (w/v) glycerol, suitably, about 0.35% (w/v) glycerol, moresuitably less than 0.2% (w/v) glycerol.

There is also disclosed the use of the dried composition describedherein for preparing a dried flap endonuclease-containing composition.

There is also disclosed the use of the dried composition describedherein combined with a reconstitution solution for performing a nucleicacid based assay.

There is also disclosed the use of the dialysis composition describedherein for preparing a substantially glycerol-free flap endonucleasecomposition.

Definitions

The term “about” indicates insubstantial variation in a quantity of acomponent of a composition not having any significant effect on theactivity or stability of the composition.

A “bulking agent” provides a matrix for the deposit of proteins andother reagents during drying and storage. (Carpenter et al (2002)Rational design of stable lyophilized protein formulations. KluwerAcademic/Plenum, New York, pp. 109-133). Bulking agents can be used toform a product “cake” or other structure, and can prevent protein frombeing lost from the vial during drying and increase protein stability.

A “chelating agent” is an agent that sequesters divalent ions, such asMg²⁺ or Mn²⁺ required for enzyme activity.

The terms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.“Lyophilisate” refers to a lyphophilized substance.

The term “stringent” in reference to nucleic acid hybridization(including “stringent hybridization conditions” or “stringentconditions”) refers to conditions where a specific oligonucleotide isable to hybridize with target nucleic acids over other nucleic acidspresent in the test sample. It will be appreciated that these conditionsmay vary depending upon factors including the GC content and length ofthe oligonucleotide, the hybridization temperature, the composition ofthe hybridization reagent or solution, and the degree of hybridizationspecificity sought. Appropriate hybridization conditions are well knownin the art for probes, oligonucleotides, target captureoligonucleotides, blockers and other oligonucleotides, may be predictedbased on sequence composition, or can be determined by using routinetesting methods (e.g., Sambrook et al., Molecular Cloning. A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

The term “cleavage structure” refers to a structure that is formed bythe interaction of a number of nucleic acids to form a configurationwherein two of the nucleic acids each contain a nucleobase configured tohybridize at a single nucleobase position on a third nucleic acid suchthat an overlap is formed between the first two nucleic acids where theresulting non-hybridized flap region (e.g., cleavage structure) iscleavable by a flap endonuclease. The cleavage structure is a substratefor specific cleavage by the flap endonuclease, in contrast to a nucleicacid molecule that is a substrate for non-specific cleavage by agentssuch as phosphodiesterases, which cleave nucleic acid molecules withoutregard to secondary structure (i.e., no formation of a duplex structureis required). For a discussion of a cleavage structure, as well as otheraspects of a flap endonuclease detection assay, please see U.S. Pat. No.5,846,717.

A “flap endonuclease,” as used herein, refers to a class of nucleolyticenzymes that act as structure-specific 5′ endonucleases on nucleic acidstructures with a duplex containing a single-stranded 5′ overhang, orflap, on one of the strands that is displaced by another strand ofnucleic acid (i.e., such that there are overlapping nucleotides wherethe adjacent first and second probes hybridize to a target). A flapendonuclease may also be referred to as a “5′ endonuclease” or by theacronym “FEN” for short. FENs catalyze hydrolytic cleavage of thephosphodiester bond at the junction of single- and double-strandednucleic acid, releasing the overhang, or flap. FENs are reviewed byCeska and Savers (Trends Biochem. Sci. 23:331-336, 1998) and Liu et al.(Annu. Rev. Biochem. 73:589-615, 2004).

An “overlap region” or “flap region” consists of the base or bases ofthe first probe oligonucleotide (e.g., a signal probe) that hybridize tothe target and are overlapped by the second probe oligonucleotide (e.g.,an invader probe). The base on the 3′ end of the second probeoligonucleotide determines the end of the overlap region and may or maynot hybridize to the target.

A “first probe oligonucleotide,” in reference to a cleavage-based assay,refers to an oligonucleotide that interacts with a target nucleic acidto form a cleavage structure (e.g. a signal probe) in the presence of a“second probe oligonucleotide” that hybridizes to a region upstream ofthe first probe oligonucleotide (e.g. an invader probe). When annealedto the target nucleic acid, the first probe oligonucleotide and targetform a cleavage structure and cleavage by a flap endonuclease can occurwithin the first probe oligonucleotide. In the presence of anoverlapping second probe oligonucleotide upstream of the first probeoligonucleotide along the target nucleic acid, the site of cleavagewithin the first probe oligonucleotide will occur after the lastoverlapping base (cleavage depends on at least one overlapping base ofthe second probe with target-hybridized bases of the first probe). Inaddition to a target-hybridizing region that hybridizes to a targetsequence within the target nucleic acid, a first probe oligonucleotidecontains a non-target-hybridizing region at the 5′ end (also referred toas a “flap region”). When first and second probe oligonucleotides areannealed to a target nucleic acid, site-specific cleavage by a flapendonuclease occurs to generate a cleavage product that contains theflap region and the overlap region of the first probe oligonucleotide.

A “second probe oligonucleotide” in reference to a cleavage-based assay,refers to an oligonucleotide that contains a sequence at its 3′ endthat, when annealed to the target nucleic acid, overlaps the 5′ end ofthe target-hybridizing sequence within a downstream first probeoligonucleotide; typically, these regions will compete for hybridizationto the same segment along a complementary target nucleic acid. Thesecond probe can, in some instances, be used as a primer as well as aninvader probe. The 3′ terminal nucleotide of the second probeoligonucleotide may or may not base pair with a nucleotide in the targetnucleic acid. In some variations, only the 3′ terminal nucleotideoverlaps the 5′ end of the target-hybridizing sequence of the firstprobe oligonucleotide. The cleaved second probe oligonucleotide can beinvolved in a secondary reaction where they act as probes on afluorescent resonance energy transfer cassette (e.g. a FRET probe orFRET cassette) leading to the formation of an overlapping structure thatis recognised by a Cleavase® enzyme. When the FRET cassette is cleaved afluorophore is released from a quencher on the FRET cassette generatinga fluorescence signal.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleavageproduct with a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

An “amplification oligomer” is a primer or promoter primer that cansupport template-dependent replication. An amplification oligomer pairis a pair of such oligomers that support template dependent replicationof opposing strands of a template. Multiplex amplification isamplification performed with multiple amplification oligomer pairssimultaneously.

A “detection probe” is an oligonucleotide that can hybridize to anamplification product or an initial target nucleic acid to revealpresence or amount of the amplification product. Such detection probesoften incorporate a molecule giving a fluorescent or other detectablesignal in which case they are referred to as detectably labelled probes.

A “primer-probe set” is a combination of primers and detection probeconfigured for generating an amplification product from a templatenucleic acid.

“Reconstitution time” is the time that is required to rehydrate a driedformulation with a solution to result in a solution that is free ofparticles or turbidity to the naked eye.

“Relative Fluorescence Units (RFU)” are a measure of unquenchedfluorophore. In nucleic acid based amplification reactions, the presenceof a product is determined by measuring RFU at a number of cycle times(Ct).

“Ct” refers is the number of cycles that was required to reach theexponential phase in a real time PCR. Ct is inversely related to theamount of analyte in a sample.

“Positivity”, when in reference to assay reaction results, refers to thepercent of samples that crossed over into the exponential threshold, ina test involving a plurality of samples. For example, when the pluralityof samples is twelve samples, and when the number of samples crossingover was determined to be six, positivity is “50%.”

A “single unit dose” or “SUD” refers to a volume of a reaction mixturethat is used to perform an assay on a single sample. A single unit dosecan be in liquid form or in dried form. By way of example, a single unitdose can be a dried pellet containing reagents useful for the detectionof a single sample in a single vessel.

“LOD” is limit of detection of an analyte. LOD+1 is the LOD that theuser has detected plus one log. In other words, LOD+1 is ten times thenumber of analytes that is the LOD.

Compositions disclosed as including dTTP and dUTP can include dTTP ordUTP or both at the concentrations indicated. Likewise when acomposition is disclosed as containing dTTP or dUTP, the disclosureshould be understood as alternatively including a composition includingboth dTTP and dUTP at the indicated concentrations.

DETAILED DESCRIPTION I. General

The present disclosure is based, at least in part, on the finding thatinstability of prior lyophilized kits for performing nucleic acid basedassay is due to the presence of inorganic salts. These salts can resultin undesired hybridization products or other by-products before, duringand after drying. These salts also make a dried composition hygroscopicsuch that it absorbs water, thus requiring limited exposure to humidity,refrigerated or deep freeze storage and/or storage in the presence of adesiccant. The presence of water and salts causes the enzyme componentof such kits to lose activity prematurely and can also facilitatehybridization of nucleic acids in such kits to each other. The presentdisclosure has overcome these problems by drying reagents for conductinga nucleic acid based assay from bulk reagents essentially free ofinorganic salts. Such salts are supplied on reconstitution of the driedcomposition. Contrary to the expectation that inorganic salts arenecessary for stability of enzymes used for nucleic acid based assays,it has been found that reaction mixtures dried essentially free ofinorganic salt can be stored long term above freezing, with full orsubstantial retention of activity on reconstitution.

II. Bulk Reagents & Dried Pellets.

Bulk reagents (sometimes referred to as prelyophilized mixtures,solutions, aqueous solutions or compositions) according to thedisclosure typically include a flap endonuclease, a bulking agent, anorganic buffer. Bulk reagents may or may not also include one or morenucleic acids and/or dNTPs. Bulk reagents may include chelators andRNase inhibitors.

Such bulk reagents are essentially free of inorganic salts meaning theconcentration of inorganic salt individually and collectively is lessthan 5 mM and preferably less than 1 mM. Preferably, the concentrationof Mg2+ is less than 1 mM, less than 0.5 mM, less than 0.1 mM or lessthan 0.05 mM. Preferably, the concentration of Na+ is less than 1 mM,less than 0.5 mM, less than 0.1 mM or less than 0.05 mM. Preferably, theconcentration of K+ is less than 1 mM, less than 0.5 mM, less than 0.1mM or less than 0.05 mM. Preferably, the concentration of Cl− is lessthan 1 mM, less than 0.5 mM, less than 0.1 mM or less than 0.05 mM.

Such bulk reagents are substantially free of glycerol. As used herein,the term “substantially free” means that glycerol is not present in anamount generally used in an enzyme preparation. Suitably, the glycerolis present in amount of less than 5% (w/v), less than 4% (w/v), lessthan 3% (w/v), less than 2% (w/v), less than 1% (w/v), less than 0.5%(w/v), less than 0.1% (w/v) or less than 0.01% (w/v). Suitably, theglycerol is present in amounts that are not detectable usingconventional methods that are known in the art. For example, the Abcam(Cambridge, UK) Glycerol Assay Kit can be used to measure free glycerolconcentration by enzymatically oxidizing glycerol to generate a productwhich reacts with a probe to generate color and fluorescence. The kitcan detect 50 pmol-10 nmol of glycerol in various samples. Suitably, theglycerol is present in a concentration of less than 50 pmol.

Nucleotides for incorporation into a reaction mixture are typicallyprovided as dNTPs. Exemplary concentrations for dNTPs are 0.1 to 0.4 mMof dATP, suitably, 0.3 to 0.4 mM dATP, suitably, 0.29 to 0.46 mM dATP;0.1 to 0.4 mM of dGTP, suitably, 0.3 to 0.4 mM dGTP, suitably, 0.29 to0.4 mM dGTP; 0.1 to 0.4 mM of dCTP, suitably, 0.3 to 0.4 mM dCTP,suitably, 0.29 to 0.4 mM dCTP; 0.1 to 0.4 mM of dTTP, suitably, 0.2 to0.3 mM dATP; and 0.1 to 0.4 mM of dUTP, suitably, suitably, 0.125 to0.234 mM dUTP. Exemplary concentrations in a 1.5× lyophilization mix orfinal reaction (i.e., after reconstitution in 1.5× volume) are asfollows.

In lyo mix (mM) In reaction (mM) dATP .375 .25 dGTP .375 .25 dCTP .375.25 dTTP .284 .189 dUTP .182 .122

Such mixtures can be customized for any type of reaction that uses aflap endonuclease. Such mixtures can be customized for any type ofreaction that uses a flap endonuclease and a polymerase.

Flap endonucleases are commercially available or can be prepared by auser. A flap endonuclease is not restricted to enzymes having solely 5′nuclease activity. For example, the flap endonuclease may be a nativeDNA polymerase having 5′ nuclease activity (e. g., Taq DNA polymerase,E. coli DNA polymerase 1) or a modified DNA polymerase having 5′nuclease activity by lacking synthetic activity (e.g., a Cleavase®enzyme).

Such mixtures can be customized for different types of amplificationincluding PCR, RT-PCR and transcription mediated amplification by thechoice of polymerase enzyme and other components.

DNA polymerase enzymes are commercially available or can be prepared bya user. One example of a polymerase enzyme is a Taq polymerasecommercially available from Qiagen (Germantown, Md., cat #201203).Another example of a Taq polymerase is commercially available as GoTaq®G2 Flexi DNA polymerase (Promega, Madison, Wis., cat # M7801). Other DNApolymerases that are commercially available include, but are not limitedto, Tth DNA polymerase (e.g., Sigma-Aldrich, St. Louis, Mo., cat#11480022001), and chimeric DNA polymerases such as Phusion®High-Fidelity DNA Polymerase (NEB, Ipswich, Mass., cat # M0530S). Alsocommercially available are hot-start DNA polymerase enzymes. Forexample, a Taq polymerase is commercially available as GoTaq® Hot StartPolymerase (Promega, cat # M5001). The GoTaq® Hot Start polymerase is anantibody mediated hot start enzyme, where the Taq polymerase is bound toan antibody that blocks polymerase activity. The blocking antibody isdenatured using high heat, thus during the initial heat step of a PCRreaction, the antibody is denatured and polymerase activity is restored.Various antibodies can be used with hot start method, for example,TAQSTART antibody (Clontech Laboratories, Mountain View, Calif., cat #R028A). Similarly, other hot start polymerase enzymes are available,including chemically-mediated hot start polymerases. Equivalentpolymerase and antibodies are available from a variety of commercialsources and, alternatively, can be prepared by the user.

Reverse transcriptase enzymes are commercially available or can beprepared by a user. Examples of commercially available reversetranscriptase include, but are not limited to, MMLV (Maloney MurineLeukemia Virus) reverse transcriptase & SuperScript® III ReverseTranscriptase (e.g., ThermoFisher Scientific, Carlsbad, Calif., cat # s28025-013 & 18080-044), MMLV RT (Sigma-Aldrich, cat # M1302), AMVReverse Transcriptase (NEB, Ipswich, Mass., cat # M0277S), and GoScript™reverse transcriptase (Promega, cat # A50003). GoScript reversetranscriptase includes a reverse transcriptase and a set of reagents forsynthesis of first-strand cDNA optimized for quantitative PCRamplification. Equivalent reverse transcriptase and reagents areavailable from various commercial sources and, alternatively, can beprepared by the user.

Exemplary concentrations for a flap endonuclease in single unit dosesare between about 0.01 μg/μl to about 1.0 μg/μl, suitably, between about0.01 μg/μl and about 0.8 μg/μl, or between about 0.01 μg/μl and about0.6 μg/μl, or between about 0.01 μg/μl and about 0.4 μg/μl, or betweenabout 0.01 μg/μl and about 0.2 μg/μl, or between about 0.01 μg/μl andabout 0.1 μg/μl. More suitably, the concentrations for a flapendonuclease in single unit doses are between about 0.02 μg/μl and about0.04 μg/μl. More suitably, the concentrations for a flap endonuclease insingle unit doses are between about 0.030 μg/μl to about 0.04 μg/μl,more suitably, between about 0.030 μg/μl to about 0.035 μg/μl. Suitably,the flap endonuclease is present in the aqueous solution at about 0.010μg/μl to about 0.050 ug/ul, suitably at about 0.012 ug/ul to 0.047ug/ul.

Exemplary concentrations for DNA polymerase enzyme in single unit dosesare 0.1-0.2 U/μl (inclusive of all whole and partial numbers therein).For example 0.14 U/μl, 0.146 U/μl and 0.1687 U/μl.

One unit of DNA polymerase is defined as the amount of enzyme requiredto catalyze the incorporation of 10 nanomoles of dNTPs intoacid-insoluble material in 30 minutes at 74 degrees C. Exemplaryconcentrations of reverse transcriptase enzyme in single unit doses are0.01 U/μl-1.0 U/μl (inclusive of all whole and partial numbers therein).One unit of reverse transcriptase is defined as the amount of enzymerequired to catalyze the transfer of 1 nmol of deoxynucleotide intoacid-precipitable material in 10 minutes at 37 degrees C.

A preferred organic buffer is MOPS or Tris.

Suitably, the MOPS buffer is present at a concentration of from about 10mM to about 20 mM in the aqueous solution, suitably at a concentrationof about 12.5 mM to about 15 mM in the aqueous solution.

Suitably, the Tris buffer is present at a concentration of from about 5mM to about 60 mM in the aqueous solution, suitably at a concentrationof from about 40 to 60 mM in the aqueous solution, suitably at aconcentration of about 50 mM in the aqueous solution, suitably at aconcentration of about 10 mM in the aqueous solution.

Alternative organic buffers that can be incorporated into bulk reagentsinclude phosphate, citrate, acetate, CHES, histidine, and Good'sbuffers, such as HEPES, MES, tricine, and glycinamide, as well as buffercombinations. Other organic buffers include succinate, citrate,gluconate, phosphate, and the like. Preferred buffers are effective in apH range from about 6.0 to about 10.0 or about 7.0 to about 9.0;suitably a pH of about 7.5 or 8.0 or 8.5 is used.

A preferred bulking agent is trehalose. Other bulking agents that arecontemplated include raffinose, sucrose, mannitol, trehalose plusmannitol, sucrose plus mannitol, sucrose plus glycine, and hydroxyethylstarch. See, Cleland et al (2001) J. Pharm. Sci. 90:310; Meyer et al(2009) Eur. J. Pharm. Sci. 38:29; Webb et al (2003) J. Pharm. Sci.92:715; Garzon Rodrigues et al (2004) J. Pharm. Sci. 93:684; Qiu et al(2012) Int. J. Pharmaceuticals. 437:51); Van Dijk-Wolthuis et al (1997)Polymer. 38:6235 6242. Hydroxyethyl starch is classified as, hetastarch,hexastarch, pentastarch, and tetrastarch (see, e.g., WO2014/099198 ofChow). The bulking agent is preferably present at a concentration offrom about 0.2 M to about 0.5 M, suitably, about 0.2 M to about 0.4 M,suitably about 0.3 M to about 0.4 M, suitably, about 0.47 M, suitably,about 0.36 M.

Bulk reagents may include one more nucleic acids—such as probeoligomers, amplification oligomers, capture probes, positive controltemplate, and negative control template and the like. Bulk reagents mayinclude one more nucleic acids for performing a non-amplification basedassay including one or more probe oligonucleotides. Bulk reagents mayinclude one more nucleic acids for performing a non-amplification basedassay and an amplification based assay including one or more probeoligonucleotides and/or one or more amplification oligomers and/or oneor more amplification oligomer pairs and/or multiple amplificationoligomer pairs.

Optional additional components of a bulk reagents include RNaseinhibitor, detergents, zwitterionic detergents, anionic detergents,cationic detergents, non-ionic detergents, surfactants, primers, probes,template, polymers, biopolymers, oligosaccharides, polysaccharides,poly-glucose, amylose, chelating agent, methyl paraben, and propylparaben. An exemplary concentration for methyl paraben is 0.01-0.024% byweight, for example about 0.016%, or alternatively, about 0.010%, about0.014%, about 0.016%, about 0.020%, about 0.024%, and any rangesbordered by these values. An exemplary concentration range of propylparaben is 0.002-0.016% or 0.008%, or alternatively, about 0.002%, about0.004%, about 0.006%, about 0.008%, about 0.010%, about 0.012%, about0.014%, about 0.016% or any range bordered by these values. One unit isdefined as the amount of RNasin® Ribonuclease Inhibitor required toinhibit the activity of 5 ng of ribonuclease A by 50%. Activity ismeasured by the inhibition of hydrolysis of cytidine 2′, 3′-cyclicmonophosphate by ribonuclease A.

Chelating agents include one or more of EDTA (ethylenediaminetetraaceticacid), EGTA (ethylene glycol tetraacetic acid), EDDS(ethylenediamine-N,N′-disuccinic acid), MGDA (methylglycinediaceticacid), and DTPA (diethylene triamine pentaacetic acid). Exemplaryconcentrations for chelating agents are from 1.0 mM-2.5 mM. In oneembodiment, the use of EDTA is preferred. Suitably, the EDTA is presentat a concentration from about 0.05 to about 0.2 mM, suitably, about 0.1mM.

RNase inhibitor proteins are native and recombinant are 50 kDa proteinsthat inhibit RNase A family and human placental RNases by noncovalentlybinding to RNases in a 1:1 ratio (Promega Corp., Madison, Wis.). See,Botella-Estrada et al (2001) Cancer Gene Ther. 8:278; Polakowski et al(1992) EXS. 61:428. Exemplary concentrations of RNase inhibitor are fromabout 0.04 U/μl to about 0.4 U/μl.

Bulk reagents can contain detergent at low concentration. Detergentsinclude ionic (cationic or anionic), non-ionic and zwitterionicdetergents available from a number of commercial vendors (e.g., GenoTechnology, Inc., St. Louis, Mo.). Examples include, but are not limitedto, lithium lauryl sulfate, amprolium hydrochloride, benzalkoniumchloride, choline p-toluenesulfonate salt, dodecyltrimethylammoniumchloride, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,ethylhexadecyldimethylammonium bromide, hexadecylpyridinium chloride,hexadecyltrimethylammonium chloride, sodium dodecyl sulfate,hexadecyltrimethylammonium p-toluenesulfonate, Luviquat™,methylbenzethonium chloride, myristyltrimethylammonium bromide,N,N′,N′-Polyoxyethylene (10)-N-tallow-1,3-diaminopropane liquid,oxyphenonium bromide, tetraheptylammonium bromide,tetrakis(decyl)ammonium bromide, tricaprylylmethylammonium chloride,Amidosulfobetaine-16, tridodecylmethylammonium chloride,trimethyloctadecylammonium bromideNonidet P-40®, Tween-20®, Tween-80®,Brij-35®, Triton X-100®.

Exemplary volumes of a bulk reagents include about 1 ul, about 5 ul,about 10 ul, about 20 ul, about 24 ul, about 50 ul, about 100 ul, about200 ul, about 300 ul, about 400 ul, about 500 ul, about 600 ul, about700 ul, about 800 ul, about 900 ul, about 1,000 ul (1 mL), about 2 mL,about 5 mL, about 10 mL, about 20 mL, about 50 mL, and so on.Reconstituted compositions can be formed at the same volume, a lowervolume, or greater volume than the bulk reagents. A lower volume can beabout 90%, about 80%, about 60%, about 40%, about 20%, about 10%, orabout 5%, relative to the bulk reagents. A greater volume can be about120%, 140%, 160%, 180%, 200% (2-fold), about 4-fold, about 6-fold, about8-fold, about 10-fold, about 20-fold, that of the bulk reagents.

A sample to be analyzed can be added to the bulk reagents either beforereconstitution, at the same time as reconstitution, or afterreconstitution. In a preferred embodiment, the entire dried compositionafter reconstitution is used for combining with sample, and here therelative volumes of reconstitution solution/sample can be, for example,about 9.9/0.1, 9.8/0.2, 9.5/0.5, 9/1, 8/2, 7/3, 6/4, 5/5, and so on.

Unless otherwise specified, concentrations of reagents in bulk reagentscan be for example, 0.0% (an omitted reagent), 0.001%, 0.004%, 0.008%,0.0012%, 0.0016%, 0.0020%, 0.0030%, 0.0040%, 0.0050%, 0.0060%, 0.0080%,0.01%, 0.02%, 0.04%, 0.06%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%,1%, 2%, 3%, 4%, 5%, and the like. Also provided are reagents that are at“about” the above concentrations, less than the above concentrations,more than the above concentrations, ranges involving any two of theabove concentrations.

An exemplary bulk reagent composition is substantially free of glycerol,has an inorganic salt concentration of 5 mM or less and has 0.1-0.4 mMand suitably about 0.3 mM of each of dATP, dGTP dCTP and dUTP or dTTPand 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.03 μg/μlflap endonuclease. Some compositions include a bulking agent, suitablytrehalose, at about 0.2 M to about 0.4 M, suitably about 0.3 M. Somecompositions include a buffer, suitably MOPS buffer, suitably at a pH ofabout 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP dCTP andabout 0.182 mM dUTP or about 0.284 mM of dTTP, 0.02 μg/μl-0.40 ug/μLflap endonuclease, suitably about 0.03 μg/μl flap endonuclease, and 0.1U/μl to 0.2 U/μl, suitably, 0.146 U/μl polymerase. Some compositionsinclude a bulking agent, suitably trehalose, at about 0.2 M to about 0.4M, suitably about 0.3 M. Some compositions include a buffer, suitablyMOPS buffer, suitably at a pH of about 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP dCTP andabout 0.182 mM dUTP or about 0.284 mM of dTTP, 0.02 μg/μl-0.40 ug/μLflap endonuclease, suitably about 0.035 μg/μl flap endonuclease, 0.1U/μl to 0.2 U/μl, suitably, 0.146 U/μl polymerase, a bulking agent,suitably trehalose, at about 0.2 M to about 0.4 M, suitably about 0.3 Mand a buffer, suitably MOPS buffer, suitably at a pH of about 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP dCTP andabout 0.182 mM dUTP or about 0.284 mM of dTTP and 0.02 μg/μl-0.40 ug/μLflap endonuclease, suitably about 0.03 μg/μl flap endonuclease. Somecompositions include a bulking agent, suitably trehalose, at about 0.2 Mto about 0.4 M, suitably about 0.3 M. Some compositions include aglobular protein, suitably, bovine serum albumin (BSA), more suitably,ultrapure non-acetylated BSA. The globular protein can be present in anamount of 0.40 to 0.60 μg/μl, suitably, 0.50 μg/μl. Some compositionsinclude a buffer, suitably Tris buffer, suitably at a pH of about 8.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP dCTP andabout 0.182 mM dUTP or about 0.284 mM of dTTP and 0.02 μg/μl-0.40 ug/μLflap endonuclease, suitably about 0.03 μg/μl flap endonuclease and 0.1U/μl to 0.2 U/μl, suitably, 0.146 U/μl polymerase. Some compositionsinclude a bulking agent, suitably trehalose, at about 0.2 M to about 0.4M, suitably about 0.3 M. Some compositions include a globular protein,suitably, bovine serum albumin (BSA), more suitably, ultrapurenon-acetylated BSA. The globular protein can be present in an amount of0.40 to 0.60 μg/μl, suitably, 0.50 μg/μl. Some compositions include abuffer, suitably Tris buffer, suitably at a pH of about 8.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably 0.3 mM of each of dATP, dGTP dCTP and dUTP ordTTP and 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.03μg/μl flap endonuclease and 0.1 U/μl to 0.2 U/μl, suitably, 0.146 U/μlpolymerase, a bulking agent, suitably trehalose, at about 0.2 M to about0.4 M, suitably about 0.3 M, a globular protein, suitably, bovine serumalbumin (BSA), more suitably, ultrapure non-acetylated BSA, saidglobular protein being present in an amount of 0.40 to 0.60 μg/μl,suitably, 0.50 μg/μl, and a buffer, suitably Tris buffer, suitably at apH of about 8.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably 0.375 mM of each of dATP, dGTP dCTP and dUTP ordTTP and 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.035μg/μl flap endonuclease. In some compositions, the flap endonuclease isa Cleavase® enzyme. Some compositions include a bulking agent, suitablytrehalose, at about 0.2 M to about 0.4 M, suitably about 0.36 M. Somecompositions include a globular protein, suitably, bovine serum albumin(BSA), more suitably, ultrapure non-acetylated BSA. The globular proteincan be present in an amount of 0.40 to 0.60 μg/μl, suitably, 0.50 μg/μl.Some compositions include a buffer, suitably MOPS buffer, suitably at apH of about 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably 0.375 mM of each of dATP, dGTP dCTP and dUTP ordTTP and 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.035μg/μl flap endonuclease and 0.1 U/μl to 0.2 U/μl, suitably, 0.167 U/μlpolymerase. Some compositions include a bulking agent, suitablytrehalose, at about 0.2 M to about 0.4 M, suitably about 0.36 M. Somecompositions include a globular protein, suitably, bovine serum albumin(BSA), more suitably, ultrapure non-acetylated BSA. The globular proteincan be present in an amount of 0.40 to 0.60 μg/μl, suitably, 0.50 μg/μl.Some compositions include a buffer, suitably MOPS buffer, suitably at apH of about 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably 0.375 mM of each of dATP, dGTP dCTP and dUTP ordTTP and 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.035μg/μl flap endonuclease and 0.1 U/μl to 0.2 U/μl, suitably, 0.167 U/μlpolymerase, a bulking agent, suitably trehalose, at about 0.2 M to about0.4 M, suitably about 0.36 M, a globular protein, suitably, bovine serumalbumin (BSA), more suitably, ultrapure non-acetylated BSA, saidglobular protein being present in an amount of 0.40 to 0.60 μg/μl,suitably, 0.50 μg/μl, and a buffer, suitably MOPS buffer (such as 15 mMMOPS buffer), suitably at a pH of about 7.5.

Another exemplary bulk reagent composition is substantially free ofglycerol, has an inorganic salt concentration of 5 mM or less and has0.1-0.4 mM and suitably 0.375 mM of each of dATP, dGTP dCTP and dUTP ordTTP and 0.02 μg/μl-0.40 ug/μL flap endonuclease, suitably about 0.035μg/μl flap endonuclease and 0.1 U/μl to 0.2 U/μl, suitably, 0.169 U/μlpolymerase, a bulking agent, suitably trehalose, at about 0.2 M to about0.4 M, suitably about 0.36 M, a globular protein, suitably, bovine serumalbumin (BSA), more suitably, ultrapure non-acetylated BSA, saidglobular protein being present in an amount of 0.40 to 0.60 μg/μl,suitably, 0.51 μg/μl, a buffer, suitably MOPS buffer (such as 15 mM MOPSbuffer), suitably at a pH of about 7.5 and a chelating agent, suitablyEDTA at a concentration from about 0.05 to 0.2 mM, suitably, about 0.15mM.

After formation of a bulk reagent composition it may be left at roomtemperature for a significant period before drying. The period can befor up to 8 hr before the drying step is initiated, or alternatively,for up to 1 hr, up to 2 hr, up to 4 hr, up to 6 hr, up to 10 hr, up to12 hr, up to 14 hr, before the drying step is initiated. Inclusion ofsalts in the bulk reagent results in undesired hybridization productsand other by-products during this incubation period. Such undesiredhybridization and by-products are reduced or eliminated by forming thebulk reagent composition essentially without inorganic salt.

The presence of inorganic salts in a bulk reagent can result in one ormore of the following undesirable properties. Nucleic acids mayhybridize together, the hybridization being stimulated by the presenceof inorganic salts such as potassium, sodium, manganese, magnesiumand/or chloride. Also in the presence of inorganic salts like manganeseand magnesium, undesirable enzyme activity can occur. As a result ofnucleic acid hybridization and enzyme activity in the presence of salt,undesired side-products may start to form. Additionally, inorganic saltsare hygroscopic and will draw moisture into a dried pellet. Rehydrationof the dried pellet reduces storage stability, enzyme stability, andallows for additional spurious side product formation.

A dried pellet can contain regents to provide one single unit dose(SUD), or optionally, two or more SUDs. A single unit dose is acollection of regents necessary to perform a nucleic acid based assay onnor more than a single sample. Single unit dose can refer to a liquidreagent or a dried pellet. It is notable that a single unit dose, asreferred to herein, need not contain all of the reagents necessary toperform a nucleic acid based assay on a single sample. A single unitdose may lack a reagent needed for performing nucleic acid based assayreactions. Similarly, a single unit dose may contain an insufficientamount of a reagent for performing nucleic acid based assay reactions.By way of example only, a dried single unit dose pellet may compriseadequate units of flap endonuclease for performing a nucleic acid basedassay, but may contain no magnesium. In an example such as this, themagnesium can be added to the dried single unit dose pellet, such as byway of a reconstitution solution. Also by way of example only, a driedsingle unit dose may comprise an inadequate amount of dNTPs forperforming a nucleic acid based assay. In an example such as this, theremainder of the dNTPs can be added to the dried single unit dosepellet, such as by way of a reconstitution solution. Ordinarily skilledartisans in possession of this disclosure will readily generate SUDs anddried pellet SUDs with varied compositions, as these examples arenon-limiting.

In a preferred embodiment, bulk reagent comprises 5 mM or less ofinorganic salt content, more preferably 4 mM or less of inorganic saltcontent, more preferably 3 mM or less of inorganic salt content, morepreferably 2 mM or less of inorganic salt content, more preferably 1 mMor less of inorganic salt content, or more preferably 500 uM or less ofinorganic salt content. Thus a preferred concentration range ofinorganic salt in a bulk reagent is from about 5 mM to 0 mM inorganicsalt content (inclusive of all whole and partial values therein). Commoninorganic salts for a nucleic acid based assay reaction mixtures includeone or more of sodium, potassium, manganese, magnesium and chloride, toname a few.

In one aspect, a bulk reagent comprises 5 mM or less of inorganic salt,the inorganic salts are present in a mass per microliter of 0.373 μg/μlor less, or 0.332 μg/μl or less, or 0.292 μg/μl or less. In a furtheraspect, a bulk reagent comprises 5 mM or less of inorganic salt, and thesodium chloride is present at a mass per microliter of 0.292 μg/μl orless, of 0.146 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 5 mM or less of inorganic salt, and the sodium ispresent at a mass per microliter of 0.115 μg/μl or less, of 0.057 μg/μlor less, or of 0.0 μg/μl. In a further aspect, a bulk reagent comprises5 mM or less of inorganic salt, and the potassium chloride is present ata mass per microliter of 0.373 μg/μl or less, of 0.186 μg/μl or less, orof 0.0 μg/μl. In a further aspect, a bulk reagent comprises 5 mM or lessof inorganic salt, and the potassium is present at a mass per microliterof 0.196 μg/μl or less, of 0.098 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 5 mM or less of inorganic salt,and the chloride is present at a mass per microliter of 0.355 μg/μl orless, of 0.178 μg/μl or less, of 0.089 μg/μl or less, or of 0.0 μg/μl.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising 5 mM or less of an inorganic salt, and the percentmass of the inorganic salt to the mass of the pellet is 0.311% or less,0.277% or less, or 0.244% or less. In a further aspect, there isprovided a vessel that contains a dried single unit dose pellet with apercent mass of inorganic salt to mass of pellet of 0.311% or less,0.277% or less, or 0.244% or less. In a further aspect, there isprovided a multiwell plate comprising one or more wells, wherein each ofthe one or more wells contains a dried single unit dose pellet thatcontains a percent mass of inorganic salt to mass of pellet of 0.311% orless, 0.277% or less, or 0.244% or less.

In one aspect, a bulk reagent comprises 4 mM or less of inorganic salt,the inorganic salts are present in a mass per microliter of 0.298 μg/μlor less, or 0.266 μg/μl or less, or 0.234 μg/μl or less. In a furtheraspect, a bulk reagent comprises 4 mM or less of inorganic salt, and thesodium chloride is present at a mass per microliter of 0.234 μg/μl orless, of 0.117 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 4 mM or less of inorganic salt, and the sodium ispresent at a mass per microliter of 0.092 μg/μl or less, of 0.046 μg/μlor less, or of 0.0 μg/μl. In a further aspect, a bulk reagent comprises4 mM or less of inorganic salt, and the potassium chloride is present ata mass per microliter of 0.298 μg/μl or less, of 0.149 μg/μl or less, orof 0.0 μg/μl. In a further aspect, a bulk reagent comprises 4 mM or lessof inorganic salt, and the potassium is present at a mass per microliterof 0.156 μg/μl or less, of 0.078 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 4 mM or less of inorganic salt,and the chloride is present at a mass per microliter of 0.284 μg/μl orless, of 0.142 μg/μl or less, of 0.071 μg/μl or less, or of 0.0 μg/μl.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising 4 mM or less of an inorganic salt, and the percentmass of the inorganic salt to the mass of the pellet is 0.249% or less,0.222% or less, or 0.195% or less. In a further aspect, there isprovided a vessel that contains a dried single unit dose pellet with apercent mass of inorganic salt to mass of pellet of 0.249% or less,0.222% or less, or 0.195% or less. In a further aspect, there isprovided a multiwell plate comprising one or more wells, wherein each ofthe one or more wells contains a dried single unit dose pellet thatcontains a percent mass of inorganic salt to mass of pellet of 0.249% orless, 0.222% or less, or 0.195% or less.

In one aspect, a bulk reagent comprises 3 mM or less of inorganic salt,the inorganic salts are present in a mass per microliter of 0.224 μg/μlor less, or 0.199 μg/μl or less, or 0.175 μg/μl or less. In a furtheraspect, a bulk reagent comprises 3 mM or less of inorganic salt, and thesodium chloride is present at a mass per microliter of 0.175 μg/μl orless, of 0.088 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 3 mM or less of inorganic salt, and the sodium ispresent at a mass per microliter of 0.069 μg/μl or less, of 0.034 μg/μlor less, or of 0.0 μg/μl. In a further aspect, a bulk reagent comprises3 mM or less of inorganic salt, and the potassium chloride is present ata mass per microliter of 0.224 μg/μl or less, of 0.112 μg/μl or less, orof 0.0 μg/μl. In a further aspect, a bulk reagent comprises 3 mM or lessof inorganic salt, and the potassium is present at a mass per microliterof 0.117 μg/μl or less, of 0.059 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 3 mM or less of inorganic salt,and the chloride is present at a mass per microliter of 0.213 μg/μl orless, of 0.107 μg/μl or less, of 0.053 μg/μl or less, or of 0.0 μg/μl.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising 3 mM or less of an inorganic salt, and the percentmass of the inorganic salt to the mass of the pellet is 0.186% or less,0.166% or less, or 0.146% or less. In a further aspect, there isprovided a vessel that contains a dried single unit dose pellet with apercent mass of inorganic salt to mass of pellet of 0.186% or less,0.166% or less, or 0.146% or less. In a further aspect, there isprovided a multiwell plate comprising one or more wells, wherein each ofthe one or more wells contains a dried single unit dose pellet thatcontains a percent mass of inorganic salt to mass of pellet of 0.186% orless, 0.166% or less, or 0.146% or less.

In one aspect, a bulk reagent comprises 2 mM or less of inorganic salt,the inorganic salts are present in a mass per microliter of 0.149 μg/μlor less, or 0.133 μg/μl or less, or 0.117 μg/μl or less. In a furtheraspect, a bulk reagent comprises 2 mM or less of inorganic salt, and thesodium chloride is present at a mass per microliter of 0.117 μg/μl orless, of 0.058 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 2 mM or less of inorganic salt, and the sodium ispresent at a mass per microliter of 0.046 μg/μl or less, of 0.023 μg/μlor less, or of 0.0 μg/μl. In a further aspect, a bulk reagent comprises2 mM or less of inorganic salt, and the potassium chloride is present ata mass per microliter of 0.149 μg/μl or less, of 0.075 μg/μl or less, orof 0.0 μg/μl. In a further aspect, a bulk reagent comprises 2 mM or lessof inorganic salt, and the potassium is present at a mass per microliterof 0.078 μg/μl or less, of 0.039 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 2 mM or less of inorganic salt,and the chloride is present at a mass per microliter of 0.142 μg/μl orless, of 0.071 μg/μl or less, of 0.036 μg/μl or less, or of 0.0 μg/μl.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising 2 mM or less of an inorganic salt, and the percentmass of the inorganic salt to the mass of the pellet is 0.124% or less,0.111% or less, or 0.097% or less. In a further aspect, there isprovided a vessel that contains a dried single unit dose pellet with apercent mass of inorganic salt to mass of pellet of 0.124% or less,0.111% or less, or 0.097% or less. In a further aspect, there isprovided a multiwell plate comprising one or more wells, wherein each ofthe one or more wells contains a dried single unit dose pellet thatcontains a percent mass of inorganic salt to mass of pellet of 0.124% orless, 0.111% or less, or 0.097% or less.

In one aspect, a bulk reagent comprises 1 mM or less of inorganic salt,the inorganic salts are present in a mass per microliter of 0.075 μg/μlor less, or 0.066 μg/μl or less, or 0.058 μg/μl or less. In a furtheraspect, a bulk reagent comprises 1 mM or less of inorganic salt, and thesodium chloride is present at a mass per microliter of 0.058 μg/μl orless, of 0.029 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 1 mM or less of inorganic salt, and the sodium ispresent at a mass per microliter of 0.023 μg/μl or less, of 0.011 μg/μlor less, or of 0.0 μg/μl. In a further aspect, a bulk reagent comprises1 mM or less of inorganic salt, and the potassium chloride is present ata mass per microliter of 0.075 μg/μl or less, of 0.037 μg/μl or less, orof 0.0 μg/μl. In a further aspect, a bulk reagent comprises 1 mM or lessof inorganic salt, and the potassium is present at a mass per microliterof 0.039 μg/μl or less, of 0.020 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 1 mM or less of inorganic salt,and the chloride is present at a mass per microliter of 0.071 μg/μl orless, of 0.036 μg/μl or less, of 0.018 μg/μl or less, or of 0.0 μg/μl.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising 1 mM or less of an inorganic salt, and the percentmass of the inorganic salt to the mass of the pellet is 0.062% or less,0.055% or less, or 0.049% or less. In a further aspect, there isprovided a vessel that contains a dried single unit dose pellet with apercent mass of inorganic salt to mass of pellet of 0.062% or less,0.055% or less, or 0.049% or less. In a further aspect, there isprovided a multiwell plate comprising one or more wells, wherein each ofthe one or more wells contains a dried single unit dose pellet thatcontains a percent mass of inorganic salt to mass of pellet of 0.062% orless, 0.055% or less, or 0.049% or less.

In one aspect, a bulk reagent comprises 500 uM or less of inorganicsalt, the inorganic salts are present in a mass per microliter of 0.037μg/μl or less, or 0.033 μg/μl or less, or 0.029 μg/μl or less. In afurther aspect, a bulk reagent comprises 500 uM or less of inorganicsalt, and the sodium chloride is present at a mass per microliter of0.029 μg/μl or less, of 0.015 μg/μl or less, or of 0.0 μg/μl. In afurther aspect, a bulk reagent comprises 500 uM or less of inorganicsalt, and the sodium is present at a mass per microliter of 0.011 μg/μlor less, of 0.006 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 500 uM or less of inorganic salt, and thepotassium chloride is present at a mass per microliter of 0.037 μg/μl orless, of 0.019 μg/μl or less, or of 0.0 μg/μl. In a further aspect, abulk reagent comprises 500 uM or less of inorganic salt, and thepotassium is present at a mass per microliter of 0.020 μg/μl or less, of0.010 μg/μl or less, or of 0.0 μg/μl. In a further aspect, a bulkreagent comprises 500 uM or less of inorganic salt, and the chloride ispresent at a mass per microliter of 0.036 μg/μl or less, of 0.018 μg/μlor less, of 0.009 μg/μl or less, or of 0.0 μg/μl. In a further aspect, adried pellet is made from drying a liquid bulk reagent comprising 500 uMor less of an inorganic salt, and the percent mass of the inorganic saltto the mass of the pellet is 0.031% or less, 0.028% or less, or 0.024%or less. In a further aspect, there is provided a vessel that contains adried single unit dose pellet with a percent mass of inorganic salt tomass of pellet of 0.031% or less, 0.028% or less, or 0.024% or less. Ina further aspect, there is provided a multiwell plate comprising one ormore wells, wherein each of the one or more wells contains a driedsingle unit dose pellet that contains a percent mass of inorganic saltto mass of pellet of 0.031% or less, 0.028% or less, or 0.024% or less.

In one aspect, a bulk reagent comprises from about 5 mM to about 500 uMof inorganic salt, the inorganic salts are present in a mass permicroliter from about 0.373 μg/μl to about 0.029 μg/μl. In a furtheraspect, a bulk reagent from about 5 mM to about 500 uM of inorganicsalt, and the sodium chloride is present at a mass per microliter 0.292μg/μl to about 0.029 μg/μl. In a further aspect, a bulk reagent fromabout 5 mM to about 500 uM of inorganic salt, and the sodium is presentat a mass per microliter 0.115 μg/μl to about 0.006 μg/μl. In a furtheraspect, a bulk reagent comprises from about 5 mM to about 500 uM ofinorganic salt, and the potassium chloride is present at a mass permicroliter from about 0.373 μg/μl to about 0.019 μg/μl. In a furtheraspect, a bulk reagent from about 5 mM to about 500 uM of inorganicsalt, and the potassium is present at a mass per microliter 0.196 μg/μlto about 0.010 μg/μl. In a further aspect, a bulk reagent from about 5mM to about 500 uM of inorganic salt, and the chloride is present at amass per microliter 0.355 μg/μl to about 0.009 μg/μl. In a furtheraspect, a bulk reagent comprises from about 5 mM to about 500 uM ofinorganic salt, the inorganic salts are present in a mass per microliterfrom about 0.373 μg/μl to about 0.029 μg/μl, and the sodium chloride ispresent at a mass per microliter of about 0 μg/μl. In a further aspect,a bulk reagent comprises from about 5 mM to about 500 uM of inorganicsalt, the inorganic salts are present in a mass per microliter fromabout 0.373 μg/μl to about 0.029 μg/μl, and the potassium chloride ispresent at a mass per microliter of about 0 μg/μl. In a further aspect,a dried pellet is made from drying a liquid bulk reagent comprising from5 mM to 500 uM of an inorganic salt, and the percent mass of theinorganic salt to the mass of the pellet is from about 0.311% to 0.024%.In a further aspect, a dried pellet is made from drying a liquid bulkreagent comprising from 5 mM to 500 uM of an inorganic salt, and thepercent mass of the inorganic salt to the mass of the pellet is fromabout 0.311% to 0.024%, and the percent mass of the sodium chloride tomass of the pellet is about 0%, or the percent mass of the potassiumchloride to mass of the pellet is about 0%. In a further aspect, thereis provided a vessel that contains a dried single unit dose pellet witha percent mass of inorganic salt to mass of pellet is from about 0.311%to 0.024%, and the percent mass of the sodium chloride to mass of thepellet is about 0% or the percent mass of the potassium chloride to massof the pellet is about 0%. In a further aspect, there is provided amultiwell plate comprising one or more wells, wherein each of the one ormore wells contains a dried single unit dose pellet that contains apercent mass of inorganic salt to mass of pellet is from about 0.311% to0.024%, and the percent mass of the sodium chloride to mass of thepellet is about 0% or the percent mass of the potassium chloride to massof the pellet is about 0%.

In one embodiment there is provided a multiwell plate comprising one ormore wells. In one aspect, one or more wells comprise walls that areconstructed from a material comprising a low moisture-vapor transmissionrate, thermal conductivity, optical transparency, low autofluorescence,or a combination thereof. In one aspect, a one or more wells comprisewalls that are cone shaped. In one aspect, the wells comprise wallsconfigured to fit into a thermally conductive tube receiving area of adevice for performing a nucleic acid based assay reaction. In oneaspect, one or more wells of the multiwell plate comprise an opening foraccess to the chamber of the well. In one aspect, one or more wells eachcomprises a cap to seal the opening of the associated well. In oneaspect, an opening of each of the one or more wells is sealed with a capthat is a low moisture-vapor transmission rate foil. In one aspect, anopening of each of the one or more wells is sealed with a cap that is alow moisture-vapor transmission rate elastomeric substance. In oneaspect, a multiwell plate comprises one or more wells as describedherein, and wherein a chamber of the well contains a dried single unitdose pellet comprising a flap endonuclease and an inorganic salt,wherein the percent mass of the inorganic salt to the mass of the pelletis from about 0.311% to 0.024%.

III. Equipment and Methods for Drying

Bulk reagents can be can be lyophilized using standard methods andequipment. Freeze driers are available from, e.g., GEA ProcessEngineering, Columbia, Md. Contract freeze drying services are providedby, e.g., Biopharma Technology Ltd., Winchester, Hampshire, GreatBritain and by BioPharma Solutions Sterile Contract Manufacturing,Baxter Healthcare Corp, Deerfield, Ill. Guidance for lyophilization isavailable from, e.g., L. Rey, J. C. May (eds.) (2010) FreezeDrying/Lyophilization of Pharmaceuticals and Biological Products,3^(rd). ed. Informa Healthcare, NY or Methods in Enzymology, Vol. 22,Pages 33-39, Academic Press, New York (1971); or Freeze-Drying, E. W.Flosdorf, Rheinhold, N.Y. (1949). Optionally, oxygen content can bereduced during freeze-drying (Phillips et al (2001) Biologics. 19:219).

A variety of containers are suitable for drying. A container should beable to withstand the outside pressure when the container is sealed andstored under partial vacuum. The container should be made of a materialthat allows a reasonable transfer of heat from outside to inside. Thesize of the container is preferably such that the solution to be driedoccupies not more than 20% of the total volume to avoid overflow.

Samples can be dried in separate vessels or in multi-specimen vessels. Amulti-specimen vessel means a contiguous vessel that can contain atleast two specimens such that they can be stored and manipulated inparallel but separately. Standard formats for multi-specimen receptaclesinclude 6, 24, 96, 384 or 1536 wells. The volume of each well in anexample of a 96 well format is about 300-400 microliters with a workingvolume of about 75-200 microliters. Volumes generally vary inverselywith the number of wells, typically in a range between 1 nL and 10 mLfor each well, although other sizes are also contemplated. Exemplarywells can have flat bottoms, round bottoms, or V-shaped bottoms amongothers. As used herein, a vessel is also referred to as a well. As usedherein, a multi-specimen vessel is also referred to as a multiwellplate. In addition, wells are sometimes further referred to as reactionwells. The term reaction well does not require that any reactionactually take place in the reaction well. Rather, the term is used torefer to a vessel or well that contains a reagent, and that may have noreaction therein, a partial reaction therein, or a full reactiontherein.

A multiwell plate, in some embodiments herein, can undergolyophilization to form a dried composition from an aqueous solution.Lyophilization may occur in a nest device (see copending applicationU.S. Provisional Application No. 62/200,370). A nest is a container forthe cartridge with vents which can be closed by a mechanism operablefrom outside a sealed lyophilization chamber. The nest containing themultiwell plate is placed within a lyophilization chamber with the oneor more vents in the open position. The chamber is then sealed and alyophilization atmosphere is applied throughout the chamber includingthe space within the nest. The one or more vents are then closed,thereby sealing the nest. The seal on the lyophilization chamber islater released and the nest containing the multiwell plate is removed.The nest may then be relocated and stored with the multiwell platepositioned therein until an operator is ready to use the lyophilizedcomposition located therein or to reseal the multiwell plate containingthe lyophilized specimens for further storage or sale. The wells of themultiwell plate can then be sealed substantially inhibiting entry ofmoisture from ambient air. The small amount of moisture entry into asealed multiwell plate can be prevented by storing the sealed multiwellplate in a pouch containing desiccant. Similarly, separate vessels canundergo lyophilization, and can undergo lyophilization in a nest.

Other drying methods include spray drying, fluidized bed drying,dehumidifiers, and batch contact drying where a filter cake is dried atlow temperature under vacuum to a free flowing dry product (N PCheremisinoff (2000) Handbook of Chemical Processing Equipment,Butterworth Heinemann, Boston, Mass.). Dehumidifies are available fromBry Air, Inc., Sunbury, Ohio, and DST Seibu Giken, Wyomissing, Pa.Rotary dryers, conical dryers, and shelf dryers are available (McGillAirPressure LLC, Columbus, Ohio). In one embodiment, vacuum dryersremove moisture by exposing the materials to reduced pressure, wherejust enough heat is used to replace that lost through vaporization.Desiccants include silica gel desiccants, molecular sieve desiccantssuch as aluminosilicate and synthetic zeolite, and bentonite desiccants.

Reaction mixtures are preferably dried in the same vessel as that inwhich they will be reconstituted for use.

A lyophilized or otherwise dried formulation has a low water content,for example, under 5% water by weight, under 4%, under 3%, under 2%,under 1.0%, under 0.5%, under 0.2%, under 0.1%, under 0.05%, under0.02%, under 0.01% by weight, and so on.

IV. Storage

Lyophilized or otherwise dried compositions are subject to storagebefore use. The period of storage can include a period of time in whichthe dried compositions are stored at room temperature exposed to ambientair. Such a period can be up to 3 hours, or alternatively, for up to 1.0hour, up to 1.5 hours, up to 2.0 hours, up to 2.5 hours or ranges of anyof the times, such as from 1 minute to 180 minutes, or from 1 minute to100 minutes, or from 1 minute to 60 minutes, or from 1 minute to 30minutes or from 1 minute to 20 minutes, or from 1 minute to 10 minutes.The absolute humidity during such storage can be at least 2.3 g waterper cubic meter of air at 30 degrees C., or alternatively, greater than1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 grams of water per cubic meter of airat 30 degrees C.

Storage can also include a longer period in which dried compositions aresealed substantially preventing contact with ambient air outside theseal. This period of storage can be for a long time, for example, atleast a week, at least a month, at least six months, at least a year orat least two years. A period from one month to two years is exemplary.

Ranges of storage temperatures, for long-term storage or for long orshort-term stability studies include, e.g., 0-2 degrees C., 0-4 degreesC., 2-4 degrees C., 2-6 degrees C., 20 degrees C., 25 degrees C., 30degrees C., 40 degrees C., 50 degrees C., 60 degrees C., as well assubzero temperatures such as −4 to −2 degrees C., −6 to −2 degrees C.,−8 to −2 degrees C., −10 to −2 degrees C., −20 degrees C., −40 degreesC., −60 degrees C., −80 degrees C., under liquid nitrogen. Preferably,storage is above freezing point and in the range of about 4-8 degrees C.Accelerated degradation studies can be conducted at about 25 degrees C.,about 30 degrees C., about 35 degrees C., about 40 degrees C., for aperiod of, for example, one hour, two hours, four hours, 24 hours, twodays, four days, eight days, one month, and so on. Conditions forstorage or, alternatively, for stability can testing, can be those thatfluctuate in temperature, such as those that fluctuate from above tobelow a freezing point.

The essential absence of inorganic salts reduces loss of enzyme activityand formation of by-products during storage of bulk reagents beforedrying, during short term storage of dried composition before sealing,and long term storage after sealing. Preferably enzyme activity afterall storage is at least 99% the value before immediately prior toinitiating storage, at least 98%, at least 95%, at least 90%, at least80%, at least 75%, at least 70%, at least 60%, at least 50%, at least40%, at least 30%, at least 20%, and ranges bordered by thesepercentages, of the value prior to initiating storage, or alternatively,to the value of a comparator sample stored under optimal conditions.

V. Reconstitution

A preferred reconstitution solution provides about 3.0 to about 12.0 mMMgCl₂, and about 0 to about 80 mM KCl in water. The reconstitutionsolution can also contain about 0.012 to about 0.020% (w/v) methylparaben, about 0.006 to about 0.010% (w/v) propyl paraben, and/or about0.25 to about 0.35% (v/v) absolute ethanol among other components.

Reconstitution time can be, under 1 sec, under 2 sec, under 5 sec, under10 sec, under 15 sec, under 20 sec, under 50 sec, or under 60 sec (1minute), after aqueous solution suitable for intended use of the driedcomposition is contacted with the dried composition, with contactoptionally facilitated by any of shaking, tapping vortexing, rocking,drawing in and out of a pipet tip, or folding or squeezing of amalleable vial. An exemplary reconstitution time is 2-10 sec.Reconstitution time can be measured with the reconstitution solution atany of refrigerator temperature (about 4 degrees C.), ambienttemperature solution about 23 degrees C., or with a warm solution atabout 37 degrees C. Typically, the dried composition has been removedfrom a refrigerator and is cold before addition of the reconstitutionsolution. The environment (the room) for any of these procedures istypically ambient temperature or about 23 degrees C. The time at which asubstance is determined to be reconstituted can be, for example, thetime at which the substance is determined to be completely solubilized.Complete solubilization can be determined by visual inspection, forexample, where absence of turbidity or absence of a schlieren pattern isa measure of complete solubilization. Alternatively, completesolubilization can be determined by way of an optical instrument, suchas a machine that measures light scattering.

VI. Stability of Compositions

Stability of compositions is typically assessed after reconstitution ofa dried product by determining the activity (i.e., rate or yield ofdetection) or formation of by-products that occurred in the composition.Lack of stability can result from loss of activity or formation ofby-products during storage either before or after drying. Activity orformation of by-products can be absolute or relative measures. Ifrelative, the base line for comparison can be a bulk reagent mixturebefore drying and reconstitution or a control reconstituted mixturediffering from that under test in a defined way (e.g., presence of Mg²or other salt). Activity can be assessed by rate of real time cleavageor amplification or final yield of cleavage or amplification product orhit rate. Side products can be assayed by one or more of gelelectrophoresis, a gel scanner, agarose gels, capillary electrophoresis,and so on.

The activity of a reconstituted amplification mixture (corrected ifnecessary for any differences due to a different volume ofreconstitution) is preferably within 75, 80, 85, 90, or 95% or isindistinguishable within experimental error from that of the bulkreagent before drying. The side products present within a reconstitutedamplification mixture (corrected if necessary for any differences due toa different volume of reconstitution) are preferably less than 20, 15,10, 5, 4, 3, 2 or 1% by weight or average moles of the originalcompounds present in the bulk reagent before drying. Sometimes sideproducts are below a limit of detection.

VII. Kits

The dried compositions described above can be provided in a kit. Such akit can contain the dried compositions in a vessel, such as a tube. Insome embodiments, the kit contains a multiwell plate comprising one ormore wells. Some kits contain a plurality of dried compositions suppliedin separate vessels. Some kits include one or more multiwell platesincluding multiple dried compositions in one or more sealed well membersof the multiwallmultiwell plates.

Some kits also include a reconstitution solution in a separate vesselfrom dried compositions. The reconstitution solution can be provided inbulk for dispensing aliquots into individual dried composition vesselsor can be provided in the form of one or more unit dosages, each forcombination with a single vessel containing a dried composition.

Optionally a vessel containing dried composition and a vessel containingreconstitution solution can be separated by a frangible material. Thefrangible material can be aluminium foil, polypropylene, polyester,polyvinylchloride (PVC), polyethylene. The barrier can include one, two,three or more layers, each layer having the same composition, or eachlayer having a different composition, such as a foil layer in contactwith a PVC layer. Films can be acquired from, e.g., Dow Chemical Co.,Midland, Mich. or Arkema, Inc., King of Prussia, Pa. Piercing of thefrangible material allows the reconstitution solution to contact thelyophilized composition

The kit can be designed to fit into a thermocycler or into an incubatorso that enzymatic reactions take place directly in a compartment of thekit to avoid need to transfer compositions to different reaction vesselor containers holding such vessels.

Kits can be adapted for introduction of a user-supplied reagent into avessel within the kit, for example, by way of a port, a hose, a syringepuncturing a septum (see, US2014/0121515 and US2014/0276356), oralternatively, the user-supplier reagents, such as a nucleic acidtemplate, can be mixed with reagents of the disclosure in auser-supplied container. One or more of the compartments of the kit canbe supplied in an empty state and used as a mixing chamber.

VIII. Dialysis of Flap Endonucleases

Some enzymes for use in the present disclosure are manufactured in aglycerol containing buffer. Glycerol content can impair lyophilization.It is therefore necessary to prepare solutions containing glycerol-freeenzyme solutions and substantially no inorganic salts by dialysing theenzyme into a glycerol-free buffer. This dialysis can be used tosubstantially remove the glycerol and replaces it with buffer. Thedialyzed enzyme can then be used to prepare a glycerol-freelyophilization formulation with substantially no inorganic salts.Accordingly, there is also disclosed herein a dialysis compositioncomprising, consisting or consisting essentially of an aqueous solutioncontaining an organic buffer, a bulking agent, chloride ions and achelating agent. The bulking agent can be trehalose. The bulking agentcan be present at a concentration from about 100 mM to 300 mM, suitably,200 mM. The organic buffer can be tris(hydroxymethyl)aminomethane (Tris)buffer. The Tris buffer can be present at a concentration of from 10 mMto 30 mM, suitably at a concentration of 50 mM. The chloride ions can beKCl. The chloride ions can be present at a concentration of about 40 to60 mM, suitably, 50 mM. The chelating agent can be EDTA. The chelatingagent can be present in the aqueous solution at a concentration from0.05 to 0.2 mM, suitably, 0.1 mM. The dialysis composition can comprisea flap endonuclease.

An exemplary dialysis composition is an aqueous solution comprising,consisting or consisting essentially of Tris buffer (pH 8.0), trehalose,KCl and EDTA.

Another exemplary dialysis composition is an aqueous solutioncomprising, consisting or consisting essentially of about 20 mM Trisbuffer (pH 8.0), about 200 mM trehalose, about 50 mM KCl and about 0.1mM EDTA.

A method for preparing a substantially glycerol-free flap endonucleasecomposition is also disclosed in which the flap endonuclease andglycerol is dialysed to obtain a substantially glycerol-free flapendonuclease composition. Suitably, the glycerol is present in an amountfrom about 0 to about 0.35% (w/v) glycerol, suitably, from about 0 toabout 0.2% (w/v) glycerol, suitably, about 0.1% (w/v) glycerol,suitably, about 0.01% (w/v) glycerol.

EXAMPLES Example 1 Bulk Reagents

Examples 1 to 3 illustrate making a bulk reagent, drying a single unitdose (SUD) volume of the bulk reagent to get a SUD dried pellet in avessel, and reconstituting the dried pellet to get an SUD amplificationand detection mixture. Table 1 and Table 2 disclose components ofexemplary bulk reagents for drying. The two tables also discloseexemplary single unit dose concentrations. Master mix 1 was a 2× mastermix comprising 0.4 mM of each of dATP, dGTP, dCTP and dTTP; 0.8 mM dUTP;BSA, and substantially no inorganic salts. Taq polymerase in Table 1 wasin a glycerol free Tris buffer containing a cationic detergent. 2×Master Mix 2 is: 0.4 mM dATP, dGTP, dCTP, 0.8 mM dUTP, 0.74 U/uL GoTaq®MDx Hot Start polymerase, glycerol free in proprietary buffer containingTris and non-acetylated BSA, 0.48 M trehalose. 50× GoScript RT Mix is asfollows: 20 U/μL GoScript®, 8 Units/μL RNasin® Plus RNase Inhibitor inTable 2; 10 U/μL GoScript®, 8 Units/μL RNasin™ Plus in Table 1.

GoScript® RT Custom is a concentrated solution of GoScript RT at 160U/uL, glycerol free, no RNase inhibitor. Stabilizers that can beincluded deamidation inhibitors, anti-oxidants, detergents, andsurfactants, such as the surfactants: fatty acid esters of sorbitanpolyethoxylates (e.g., polysorbate 20 or polysorbate 80), and poloxamer188.

TABLE 1 Exemplary Bulk Reagent for Drying Bulk Reagent SUD Workablerange (concentration with Quantity per (concentration with(concentration with Description appropriate units) Liter appropriateunits) appropriate units) 1.4M Trehalose 0.30M 214 mL 0.24M 0.16-0.32MSoln, EDTA 2.19 mM 4.4 mL 1.75 mM 0.0-3.5 mM 0.5M pH 8.0 2X Master Mix1, 1.25X 625 mL 1X w/o salt, w/o MgCl₂ Taq polymerase 0.4 U/μL 400 kU0.32 U/μL 0.1-1.0 U/μl (50 U/μl) (8.00 mL) 50X GoScript ™ 1.25X 25.0 mL1X RT Mix for 1-Step RT-qPCR, Low Glycerol, Custom GoScript ™ RT 0.25U/μL 250 kU 0.20 U/μL 0.1-0.6 U/μl Custom Formulation, (1.6 mL) 160 U/μL10X oligonucleotide 1.25X 125 mL 1X 0.7X-1.3X mix

TABLE 2 Exemplary Bulk Reagent for Drying Bulk Reagent SUD Workablerange (concentration with Quantity (concentration with (concentrationwith Description appropriate units) per Liter appropriate units)appropriate units) Solution EDTA 2.19 mM 4.38 mL 1.75 mM 0.0-3.5 mM 0.5MpH 8.0 2X Master Mix 2, 1.25X  625 mL 1X w/o salt, w/o MgCl₂, w/extraTaq Pol, w/trehalose, w/nucleotides from trehalose 0.30M ″ 0.24M0.16-0.32M From Taq Pol 0.46 U/μL ″ 0.37 U/μL 0.1-1.0 U/μl 50XGoScript ™ 1.25X 25.0 mL 1X RT Mix for 1-Step RT-qPCR, Low Glycerol,Custom, w/RNasin Plus From RT 0.5 U/μL ″ 0.4 U/μL 0.1-0.6 U/μl 10Xoligonucleotide 1.25X  125 mL 1X 0.7X-1.3X mix

TABLE 3 Exemplary Bulk Reagent for Drying Stock End DescriptionConcentration Concentration Final Vol AllStart 2X Master  2X 1X 12μl/reaction Mix (without KCL and withoutMgCL) 10X PnP Mix 10X 1X 2.5μl/reaction Z05 DNA Polymerase 200 U/μl  4 U/μl 1 μl/50 μl reaction mixvolume AMV Reverse 80 U/μl 5 U/μl 3.13 μl/50 μl reaction mix volumeTranscriptase T4G32P (protein for 10 U/μl 2.5 U/μl   12.50 μl/50 μlreaction mix volume unfolding nucleic acids) Enzyme Dilution 33.4 μl/50μl reaction mix volume Buffer 10X oligonucleotide 10X 1.25X   1X mix

The 10× oligonucleotide mix in each of the bulk amplification reagentsfor Tables 1 to 3 included collections of primers and probes forperforming amplification and detection reactions in the below examples.Ordinarily skilled artisans will understand how to prepare primer andprobe mixtures for an amplification reaction (see e.g., Innis, MichaelA. et al., PCR Protocols: A Guide to Methods and Applications, AcademicPress (1990)). For the below examples, primers and probes were presentin the bulk reagent at concentrations from about 8 uM to about 12 uM.

Example 2. Lyophilization

In this example, the bulk reaction mixtures were dried using alyophilizer. 24 microliters of bulk amplification reagents describedgenerally in example 1 was added to a vessel and then loaded into alyophilization chamber. For the examples herein, 24 ul represents theamount of bulk reagent used to perform an amplification and detectionreaction on a single sample (also referred to as a single unit dose oran SUD). In this example, a multiwell plate (specifically a 12-wellplate) was used for both the lyophilization reaction and for storage ofthe lyophilized (dried) pellet present in the wells of the multiwellplate. Each of the 12 wells of the 12-well plate received a 24 ulaliquot of the bulk reaction mixture. A lyophilization cycle was turnedon (about 36 hour run). Following the lyophilization cycle, the 12-wellplate was retrieved and transferred to a location where the individualvessels of the 12-well plate were sealed. Vessels were sealed with ametallic foil over the vessel opening. The metallic foil was a lowmoisture-vapor transmission rate foil. The sealed 12-well plates werethen pouched with a desiccant.

Example 3. Reconstitution Solution

This example describes one reconstitution solution. The purpose of thereconstitution solution is to rehydrate the dried pellet in preparationfor using the reconstituted pellet to perform detection nucleic acidbased reaction on a sample. To each of the 12-wells of the 12-well platefrom example 2, 24 ul of reconstitution solution was dispensed bypre-piercing the foil cover on the well and then dispensing thereconstitution solution into the well. Table 4 discloses thereconstitution solution used in these examples, providing both bulkreconstitution solution concentrations and final assay concentrations.Following reconstitution of the dried pellet, target nucleic acids froma sample were added to the wells and a PCR amplification and detectionreaction was performed.

TABLE 4 Universal Reconstitution Solution Bulk Recon Soln, Final AssaySolution (concentration with Quantity (concentration with DescriptionFormula weight appropriate units) per Liter appropriate units) MgCl₂1.00M liq Stk 5.19 mM 5.19 mls 4.15 mM KCl 74.55 g 81.3 mM 6.06   65 mMMethyl Paraben 152.15 g/mol 0.02% w/v 0.20 g .016% Propyl Paraben 180.2g/mol 0.01% w/v 0.10 g .008% Ethyl Alcohol, 46.07 g/mol 0.33% v/v 3.30mL 0.26% Absolute

Example 4. Negative Influence of Inorganic Salts on Dried Bulk Reagent

This example describes the negative influence that the presence ofinorganic salts in the bulk reagents has on the dried pellet. Two bulkreagent mixtures were prepared generally according to example 1 andTable 5. The difference between Bulk Reagent A and Bulk Reagent B inTable 5 was the presence or absence of MgCl₂ in the reaction mixtures.

TABLE 5 Bulk Reagents with and without inorganic salts Bulk Reagent ABulk Reagent B (without MgCl₂ and without KCl) (with MgCl₂ and withoutKCl) Trehalose 0.30M 0.30M Hot Start Taq DNA Polymerase 0.46 Units permicroliter 0.46 Units per microliter (glycerol free) ReverseTranscriptase 0.5 Units per microliter 0.5 Units per microliter RNasin0.2 Units per microliter 0.2 Units per microliter dNTP mix 0.25 mM dNTP,0.5 mM UTP 0.25 mM dNTP, 0.5 mM UTP Nucleic Acids^(§) 7 micromolar (uM)7 micromolar (uM) KCl 0 mM 0 mM MgCl₂ 0 mM 2.5 mM Low Glycerol Buffer2.7 mM Na⁺ 2.7 mM Na⁺ 0.035 mM K⁺ 0.035 mM K⁺ ^(§)Nucleic Acids were amultiplex primer and probe mix made up of the following primer probesets: for amplification and detection of influenza A there were 2forward primers, 3 reverse primers, and 3 probes (two different flu Atarget regions); for influenza B there were 1 forward primers, 1 reverseprimer, and 1 probe; for RSV there were 1 forward primers, 1 reverseprimer, and 1 probe for RSVA and there were 1 forward primers, 1 reverseprimer, and 1 probe for RSVB; and for the internal control there were 1forward primers, 1 reverse primer, and 1 probe. The value of 7micromolar is the sum of all primer concentrations wherein each primeris about 400 nM.

Each of liquid bulk reagents A and B were prepared on ice. Bulk reagentsA and B were then each separately aliquoted into the wells of a numberof multiwell plates (specifically here, 12-well plates were used). These12-well plates containing either 12 aliquots of bulk reagent A or 12aliquots of bulk reagent B were then separated into four differentincubation conditions: (1) 90 minute incubation on ice; (2) 90 minuteincubation at room temperature; (3) 180 minute incubation on ice; or (4)180 minute incubation at room temperature. Thus, one portion of eachbulk reagent mixture was incubated at room temperature for 180 minutesand the other portion was incubated on ice for 180 minutes. Likewise,one portion of each bulk reagent mixture was incubated at roomtemperature for 90 minutes and the other portion was incubated on icefor 90 minutes. In all cases, addition of the nucleic acid component tothe reaction mixture (added as the final component) indicated the startof the incubation times.

Following the incubation times, the aliquoted bulk reagents in the12-well plates were then lyophilized until substantially drycompositions were obtained. Each of the resulting dried compositions ina well of the 12-well plates represented a dried single unit dose for atriplex amplification and detection reaction to identify one or more ofinfluenza A, influenza B and respiratory syncytial virus B in a sample.

The dried compositions were reconstituted with a reconstitution solutioncontaining 65 mM of KCl, methyl and propyl paraben at 0.02% w/v and0.01% w/v, respectively, and 0.33% v/v ethyl alcohol absolute. Thereconstitution solution for dried compositions made from bulk reagent Aalso contained 2.5 mM MgCl₂.

All three of influenza A, influenza B, and respiratory syncytial virus Bpositive samples were combined into the reconstituted reaction mixturesat 3 times their LoD, such that all components of the reconstituted mixwere at about 80% of their bulk reagent concentration. These positivesamples were extracted viruses in a negative plasma combined with atransport medium and serially diluted to the desired concentration (withthe exception that the RSVB sample serial dilution was off by a factorof 10). As indicated in Table 6, the primers and probe mix was designedto specifically detect each of the three viral targets, namely influenzaA, influenza B, or respiratory syncytial virus type B, in a separatefluorescent channel, albeit in a single molecular reaction.

The samples were assayed using a real-time PCR compatible thermal cycler(ABI 7500FAST, Applied Biosystems, Carlsbad, Calif.). Results arepresented in Table 6. The percent positive value in the table representsthe number of samples that had RFU values that exceeded the thresholdvalue as a percentage of the 12 samples tested. The amount of virus(viral particles per assay) was an amount sufficient to give at least a95% positive compared to the positive control.

TABLE 6 Results Influenza A Influenza B RSVB Avg RFU For Avg RFU For AvgRFU For Positive Samples Positive Samples Positive Samples No. Positive/No. Positive No. Positive/ Condition % Positive /% Positive % Positive#1 Bulk Reagent B   602,587   303,112   320,017 90 min Incubation 6 of12/50% 11 of 12/92%  12 of 12/100% On Ice #2 Bulk Reagent A 1,235,7011,028,348 1,107,497 90 min Incubation 12 of 12/100% 12 of 12/100% 12 of12/100% On Ice #3 Bulk Reagent B   163,536   94,229   684,384 90 minIncubation 4 of 12/33% 11 of 12/92%  12 of 12/100% Room Temp #4 BulkReagent A 1,699,434 1,832,464 1,064,536 90 min Incubation 12 of 12/100%12 of 12/100% 12 of 12/100% Room Temp #5 Bulk Reagent B   576,226  530,568 1,048,669 180 min Incubation 12 of 12/100% 12 of 12/100% 12 of12/100% On Ice #6 Bulk Reagent A 1,396,077 1,314,236   934,325 180 minIncubation 12 of 12/100% 12 of 12/100% 12 of 12/100% On Ice #7 BulkReagent B   34,092   57,484   394,945 180 min Incubation (below RLUthreshold) 3 of 12/25% 12 of 12/100% Room Temp 0 of 12/0%  #8 BulkReagent A 1,970,034 1,896,883 1,074,004 180 min Incubation 12 of 12/100%12 of 12/100% 12 of 12/100% Room Temp

These results indicate that bulk reagents (prelyophilization solutionswithout MgCl₂ and KCl) are stable for at least 180 minutes at roomtemperature. Dried SUD pellets from bulk reagent A, once reconstitutedand combined with samples, provided amplification and detectionreactions that were more robust than those provided by dried SUD pelletsfrom bulk reagent B. Bulk reagent B, when incubated at room temperatureor even on ice for as few as 90 minutes, then dried and reconstituted togenerate an amplification reaction mixture, provided an amplificationreaction with a relatively lower signal and with an abundance of smallside products compared to bulk reagent A under the same conditions. Bulkreagents containing little to no inorganic salts are useful for dryingto generate a dried composition containing components for anamplification reaction, including polymerase enzyme components, dNTPsand nucleic acids.

Example 5. Stability of Dried Pellets, with or without Salts

This example compares the stability of single unit dose dried pelletscontaining salts with single unit dose dried pellets containing no salts(less than 6 mM inorganic salt). The single unit dose pellets were madeby drying a bulk reagent generally as described in Table 5 above.Immediately after synthesis, bulk reagent A and bulk reagent B were eachaliquoted into separate multiwell reaction plates (12-well) and driedusing a lyophilizer. Following lyophilization, the multiwell platescontaining dried pellets were placed in a nitrogen gas environmenthaving a relative humidity of about 5%, and the multiwell plates weresealed by covering the well openings with a foil. Sealed plates wereplaced into an aluminium pouch containing a desiccant, and the poucheswere then sealed. The sealed pouches containing the dried pellets inmultiwell plates were stored for eight days at 4 degrees C.

Following the eighth day, the pouched multiwell plates were transferredinto one of three conditions as follows: Condition #1-1 subset ofpouched multiwell plates containing dried pellets from bulk reagent Aand 1 subset of pouched multiwell plates containing dried pellets frombulk reagent B were removed from the pouch and placed in a 15 degrees C.environment with 70% relative humidity; Condition #2-1 subset of pouchedmultiwell plates containing dried pellets from bulk reagent A and 1subset of pouched multiwell plates containing dried pellets from bulkreagent B were removed from the pouch an placed in a 45 degrees C.environment with 15% relative humidity (accelerated stability); andCondition #3-1 subset of pouched multiwell plates containing driedpellets from bulk reagent A and 1 subset of pouched multiwell platescontaining dried pellets from bulk reagent B were placed at 4 degrees C.(the multiwell plate was in a pouch with desiccant, thus humidity waszero percent). Plates were left at these conditions for thirtyadditional days.

At the conclusion of the incubation, the dried pellets from each of theconditions were reconstituted using a reconstitution solution containing100 mM KCl and sufficient MgCl₂ for a final concentration of 2.5 mM.Reconstituted reaction mixtures were tested for amplification anddetection of an influenza A target using a real-time PCR thermal cycler(ABI PRISM 7000, Applied Biosystems, Carlsbad, Calif.). Briefly, theinfluenza A targets were extracted in a negative pool at LOD10{circumflex over ( )}(+/−1 log) along with a comparable liquidcontrol. Results are shown in Table 7.

TABLE 7 Average Total RFU (N = 4) Bulk Reagent A 1,203,798 4 .deg. C./4.deg. C. Bulk Reagent B 255,184 4 .deg. C./4 .deg. C. Bulk Reagent A1,012,184 4 .deg. C./15 .deg. C. Bulk Reagent B 261,882 4 .deg. C./15.deg. C. Bulk Reagent A 1,163,704 4 .deg. C./45 .deg. C. Bulk Reagent B382,725 4 .deg. C./45.deg. C.

These results show that single amplification reaction dried pelletscontaining less than 2.5 mM inorganic salts have higher RFU valuesfollowing storage in a number of different temperature and humidityconditions compared to single amplification reaction dried pelletscontaining 2.5 mM or more of inorganic salt.

Example 6. Lyophilization of Cleavase® Enzyme Containing Bulk Reagents

A bulk reagent containing the flap endonuclease Cleavase® wasmanufactured to contain a final glycerol concentration of 0.35%. Thisbulk reagent was aliquoted and lyophilized as generally described above.Data showed that this level of glycerol content impairs thelyophilization of the SUD pellet by causing what is referred to as “meltback” (data not presented). Melt back generally refers to the collapseof a lyophilized product. Melt back typically results from the presenceof a substance during the primary drying phase wherein the substance isdetrimental to formation of a robust lyophilized composition. Glycerolis one such substance. Therefore, a solution containing glycerol-freeCleavase® and substantially no inorganic salts was prepared by dialysingthe Cleavase® enzyme (which was stored in a 50% glycerol buffer) into aglycerol-free buffer using 20 kDa MWCO Slyde-A-Lyzer cassette system(ThermoFisher P/N #66005). The dialysis buffer composition is listed inTable 8.

TABLE 8 Cleavase ® dialysis buffer 1 composition Reagent ConcentrationTris 20 mM Trehalose 200 mM KCl 50 mM EDTA 0.1 mM pH 8.0

The dialysis of the Cleavase® enzyme in this buffer removed the glyceroland replaced it with the buffer shown in Table 8. The dialyzed Cleavase®enzyme was then used to prepare a glycerol-free bulk reagent withsubstantially no inorganic salts (Table 9). The bulk reagent was thenaliquoted as a number of SUDs into the wells of a multi-well plate andlyophilized. All SUD pellets lyophilized without exhibiting any signs ofmelt back.

TABLE 9 1.25x Pre-lyophilization formulation (version 2 formulation)Volume per Final Reagent Vendor & P/N Stock Conc. reaction concentrationGoTaq, Glycerol-free (1:10) Promega 5 U/μl working 0.67 μl 0.14 U/μlX650X stock Cleavase ® 2.0, dialyzed R&D 2.0 μg/μl 0.363 μl 0.03 μg/μl10X dNTPs R&D 2.5 mM ea 3.0 μl 0.313 mM ea 10X S. aureus Oligo mix R&D10X 3.0 μl 1X MOPS Buffer, pH 7.5 J62839 500 mM 0.6 μl 12.5 mM TrehaloseR&D 1.2M 6.0 μl 0.3M Water, MBG H20MB0106 — 10.36 μl —

-   -   The Oligo mix comprised primers for the PCR reaction, and first        probes/second probes/FRET cassettes for the cleavage based assay        reaction. The FRET cassettes were each labelled with one of the        fluorophore FAM, HEX, or ROX.

Dried compositions containing oligonucleotides for PCR amplification andcleavage-based assay detection of Staphylococcus aureus (“S. aureus”)were rehydrated with a reconstitution solution (stock reconstitutionsolution comprised 9.375 mM MgCl₂, 0.02% (w/v) methyl parapen, 0.01%(w/v) propyl paraben, 0.33% (v/v), and brought to 1 liter total volumeusing water) to generate a plurality of reaction mastermixes. S. aureustarget DNA (Positive) or nuclease free water (Negative) was added toseparate reconstituted mastermixes and topped with silicone oil. Theamplification and detection assays were performed using a Panther Fusionthermocycler. A non-lyophilized bulk reagent was tested in parallel withthe lyophilized formulations as a control.

The results in Table 10 below show that in this test the amplificationand detection system was able to successfully amplify and detect S.aureus positive samples as positive in all three channels and negativesamples as negative in all three channels and with comparable valuescompared to the wet mix control.

TABLE 10 Testing of bulk reagent version 2 - lyophilized vsnon-lyophilized control Format Sample FAM Ct FAM RFU HEX Ct HEX RFU ROXCt ROX RFU Non- Negative not 918 not 329 not 137 lyophilized detecteddetected detected control Positive 30.0 25,977 32.0 9,006 32.4 2,142Positive 29.5 29,762 31.6 10,692 32.0 2,536 Lyophilized Negative not 495not 308 not 110 detected detected detected Positive 31.1 16,924 33.24,672 34.4 1,329 Positive 30.4 19,403 32.5 5,915 33.6 1,559

Example 7. Cleavase® Pre-Lyophilization Formulation Version 3

A revised version of the S. aureus pre-lyophilization formulation wasmade that included ultrapure non-acetylated BSA and a buffer change fromMOPS buffer to Tris buffer (Table 11).

TABLE 11 1.25x pre-lyophilized formulation (version 3 formulation) using2X MMA GoTaq source Volume per Final Conc. in Reagent Vendor & P/N StockConc. pre-lyo reaction pre-lyo mix GoTaq, Glycerol-free, 2X Promega 0.74U/μl 4.75 μl 0.146 U/μl Mastermix A X991X Cleavase ® 2.0, dialyzed R&D2.2 μg/μl 0.33 μl 0.03 μg/μl BSA, Ultrapure non-acetylated * Ambion 50μg/μl 0.20 μl ~0.5 μg/μl 10X dNTPs * R&D 2.5 mM ea 2.24 μl ~0.31 mM ea10X S. aureus Oligo mix R&D 10X 3.0 μl 1X Tris Buffer, pH 8.5 * R&D 1.0M1.0 μl ~50 mM Trehalose * R&D 1.2M 4.1 μl 0.3M Water, MBG H20MB0106 —8.24 μl — * The formulation in Table 11 was supplemented with Trehalose,Tris buffer, dNTPs and non-acetyltated BSA. These reagents were added tosupplement material already included in the Promega 2X Mastermixreagent.

Promega X991× was formulated as a 2× Mastermix which when diluted 1:1would yield a reaction mix with 0.24M Trehalose, 0.2 mM dNTPs (0.4 mMdUTP), and 9.25 U GoTaq, as well as non-acetylated BSA and Tris in a 25ul reaction mixture. A total of 25 cartridges from this formulation wereprepared and successfully lyophilized.

Amplification and detection reactions were prepared and performed as isgenerally described above in Example 6. Lyophilized pellets werereconstituted using a reconstitution solution similar to that describedin Example 6, except that the concentration of MgCl₂ was increased toprovide 12.5 mM MgCl₂ in the reconstituted reaction mixture. As acontrol, a number of non-lyophilized reaction mixtures were prepared andtested. The S. aureus target nucleic acid used in these reactions was S.aureus gDNA at 100 copies/mL and at 1,000 copies/mL (approximately 3times the assay LoD for this target). In the presence of 12.5 mM MgCl₂,Tris buffered reactions showed delayed Cts, reduced RFUs and reducedT-slope values for the S. aureus positive samples. Further, the negativesamples using the Tris buffer formulation showed elevated backgroundgeneration compared to negative samples using a 10 mM MOPS bufferformulation—irrespective of the MgCl₂ concentration in the reactionmixture. Thus, based upon these results, MOPS buffer offers advantagesfor use in the bulk formulation and can be used at a concentration of upto about 15 mM in a final cartridge formulation.

The formulation illustrated in Table 11 was further varied and wastested. Formulation variations were prepared as a series of bulkreagents wherein the 11% trehalose with was substituted with varioustrehalose/sugar/polymer combinations. These bulk reagent formulationscontained the following reagent combinations and were lyophilized andtested as described above:

a) 3% Trehalose/1% 10 kDa polyvinyl propylene.

b) 3% Trehalose/2% 10 kDa polyvinyl propylene.

c) 3% Trehalose/3% 10 kDa polyvinyl propylene.

d) 3% Trehalose/1% 29 kDa polyvinyl propylene.

e) 3% Trehalose/2% 29 kDa polyvinyl propylene.

f) 3% Trehalose/3% 29 kDa polyvinyl propylene.

g) 3% Trehalose/1% 55 kDa polyvinyl propylene.

h) 3% Trehalose/2% 55 kDa polyvinyl propylene.

i) 3% Trehalose/3% 55 kDa polyvinyl propylene.

j) 3% Trehalose/1% Sucrose.

k) 3% Trehalose/2% Sucrose.

l) 3% Trehalose/3% Sucrose.

m) 11% Trehalose (Control).

Except for the condition i) and Control condition m), all combinationslyophilized well and did not show any initial collapse. Functionaltesting at t=0 also showed that there was no significant Ct delay in anyof combinations a) through m). However, when placed under acceleratedstability at 37 degrees C./95% RH, all combinations, except the controlcondition, exhibited collapse after 4 days. The control cartridge with11% trehalose did not show collapse at 37 degrees C./95% RH until 14-16days. Thus, bulk reagent formulations containing 11% are suitable forlong term storage of lyophilized pellets before use.

Example 8. Cleavase® Pre-Lyophilization Formulation Version 4

It is desirable to alter a sample input volume. Lower sample volumes areuseful to accommodate multiple tests on a patient sample. Sample inputvolumes between 210 ul and 420 ul were tested. In order to retainsensitivity at the lower sample draw volume, the eluate volume used inthe PCR reaction was increased from 5 ul to 10 ul. The extra 5 ul ofeluate sample changes the concentrations of the reagents in the reactionmixture. The reagent concentrations for the bulk reagent were adjustedto account for the different eluate volume (see Table 13).

A comparison of the 420 ul/5 ul vs 210 ul/10 ul conditions showed thatperformance was equivalent for both conditions (Table 12):

TABLE 12 420/5 μl vs 210/10 μl FAM HEX ROX CFU/mL n Pos Pos Pos % POSControl 0 12 0%  0%  0% (420/5) 100 18 0%  0% 78% 400 18 0% 11% 100% Modified 100 18 0% 11% 78% (210/10) 400 18 0% 22% 100%  Ct Control 0 12— — — (420/5) 100 18 — — 39.8 400 18 — 40.9 37.7 Modified 100 18 — 40.039.3 (210/10) 400 18 — 40.4 37.3 RFU Control 0 12 — — — (420/5) 100 18 —— 1514 400 18 — 9870 2030 Modified 100 18 — 8893 1645 (210/10) 400 18 —3780 2169

Increasing the eluate volume from 5 ul up to 10 ul resulted in a changein the SUD pellet concentration to “1.5×” in order to provide a totalreaction mixture volume of 30 ul (e.g., 20 ul mastermix+10 ul eluate).Table 13 reflects the volume change and also replaces Tris buffer with10 mM MOPS buffer, pH 7.5.

TABLE 13 1.5x S. aureus pre-lyo formulation version 4 with MOPS bufferVolume per Final Conc. in Reagent Vendor & P/N Stock Conc. pre-lyoreaction pre-lyo mix GoTaq, Glycerol-free, 2X Promega 0.74 U/μl 5.47 μl0.1687 U/μl Mastermix A X991X Cleavase ® 2.0, dialyzed R&D 1.82 μg/μl0.46 μl 0.035 μg/μl BSA, Ultrapure non-acetylated * Ambion 50 μg/μl 0.24μl ~0.509 μg/μl 10X dNTPs * R&D 2.5 mM ea 2.73 μl ~0.375 mM ea 10X S.aureus Oligo mix R&D 10X 3.6 μl 1X EDTA/EGTA, 0.1M 101068 0.1M ea 0.036μl 0.15 mM ea MOPS buffer, pH 7.5 R&D 500 mM 0.72 μl 15 mM Trehalose *R&D 1.2M   5.01 μl 0.36M Water, MBG H20MB0106 — 5.77 μl — * Thesereagents are added to supplement material already included in the 2XMastermix A reagent.

This version 4 formulation has more concentrated reagents to accommodatea doubling in sample eluate volume for the PCR reaction (5 μl 10 μl).With the change in concentrations of the formulation, a new hold timestudy was performed to ensure formulation robustness. On Day 0, a liquidS. aureus reaction mix was prepared according to Table 13 above, andsplit into 3 aliquots. The first aliquot was lyophilized immediately,the second aliquot of the mix was lyophilized after 2 days storage at2-8 degrees C., and the last aliquot was lyophilized after 4 daysstorage at 2-8 degrees C. The resulting cartridges were sealed andtested at baseline (data not shown), and also subjected to 4 weeksaccelerated stability at 30 degrees C./95% Relative Humidity (RH).Testing was carried out using MRSA cell strain at 0.5 logs above the LoD(1,000 CFU/mL), in 10 replicates per condition. The results showed thatthis formulation is able to detect MRSA at 0.5 log above the LoD anddemonstrates robustness with regards to a 0-4 days hold time prior tolyophilization, and up to 30 days of accelerated stability at 30 degreesC./95% RH after lyophilization.

Example 9

Alpha-cyclodextrin can be added to a composition to be lyophilized tocounter the inhibitory effect of a substance like SDS, which can be usedto wash captured nucleic acids. Some of the SDS will remain in thenucleic acid solution, and subsequently in the nucleic acidamplification and detection reaction. We first prepared four versions ofa formulation using S. aureus cells (GP1822) at 1,000 CFU/mL inSimulated Nasal Fluid (SNF) similar to that in Table 13, with thefollowing differences (1) 0 ug/ul cyclodextrin; (2) 0.1 ug/ulcyclodextrin; (3) 0.25 ug/ul cyclodextrin; or (4) 0.5 ug/ulcyclodextrin. The specimens were processed on the Panther FusionInstrument (P182). The 30% wash buffer was spiked into the elutionbuffer (i.e. normal WB carry over +30%). The pellets were then used intest reactions for the amplification and detection of a MRSA targetnucleic acid. No SDS was added to these amplification reaction. Thisexperiment determines if the addition of cyclodextrin to the mixtureshad an impact on a nucleic acid reaction performed with the pellet. Ascan be seen in conditions 1-4 of Table 14 the ct values weresubstantially identical for each of the test conditions, indicating thatthere is no negative impact on the performance of a pellet containing atleast as much as 0.5 ug/ul of cyclodextrin.

Following the first experiments four versions of formulation similar tothat in Table 13 were again prepared and contained one of (1) 0 ug/ulcyclodextrin; (2) 0.1 ug/ul cyclodextrin; (3) 0.25 ug/ul cyclodextrin;or (4) 0.5 ug/ul cyclodextrin. The resulting pellets were then used intest reactions for the amplification and detection of a MRSA targetnucleic acid. To this set of amplification reactions, SDS was added to afinal concentration of 30% v/v in to the reaction mix. Amplification anddetection reactions were performed. As can be seen in conditions 5-8 ofTable 14, the SDS has an inhibitory effect on the nucleic acid reaction(condition 5). 0.1 ug/ul cyclodextrin almost completely neutralized theinhibition of the SDS (condition 6), and 0.25 ug/ul and 0.5 ug/ul fullyneutralized the inhibition of the SDS (conditions 7 & 8).

TABLE 14 FAM HEX ROX RED677 cyclodextrin- Ct Std Ct Std Ct Std Ct Stdalpha conc. n n pos Avg. CT Dev. n pos Avg. Ct Dev. n pos Avg. Ct Dev. npos Avg. Ct Dev. 0% 0 ug/ul 24 24 34.22 1.12 24 34.82 0.39 24 33.75 0.4024 31.78 1.37 Wash 0.10 ug/ul 24 24 34.18 0.61 24 35.03 0.42 24 33.860.48 24 31.6 0.92 Buffer 0.25 ug/ul 24 24 33.95 0.52 24 35.08 0.31 3433.78 0.44 24 31.32 1.01 0.50 ug/ul 24 24 33.79 0.32 24 34.88 0.28 2433.73 0.39 24 31.18 0.36 30% 0 ug/ul 24 0 — — 24 39.79 1.84 24 37.972.44 20 37.86 4.30 Wash 0.10 ug/ul 24 22 36.96 1.56 24 37.73 0.92 2436.33 0.83 24 33.53 1.35 Buffer 0.25 ug/ul 24 24 35.00 2.24 24 35.430.31 24 34.56 0.30 24 32.06 1.22 0.50 ug/ul 24 24 34.63 0.69 24 35.780.92 24 34.71 0.82 24 32.88 0.99

A further data set was tested also using S. aureus cells (GP1822) at1,000 CFU/mL in Simulated Nasal Fluid (SNF). The specimens wereprocessed on the Panther Fusion Instrument (P368). The 30% wash bufferwas spiked into the elution buffer (i.e. normal WB carry over +30%).

TABLE 15 FAM HEX ROX RED677 cyclodextrin- Ct Std Ct Std Ct Std Ct Stdalpha conc. n n pos Avg. CT Dev. n pos Avg. Ct Dev. n pos Avg. Ct Dev. npos Avg. Ct Dev. 0% 0.01 ug/ul 12 12 37.77 0.96 12 38.93 0.62 12 38.380.63 12 40.48 1.37 Wash 0.05 ug/ul 12 12 37.38 0.67 12 39.08 0.52 1238.19 0.79 12 40.15 0.47 Buffer 0.10 ug/ul 12 12 37.55 0.70 12 38.650.42 12 37.93 0.48 12 39.92 0.73 0.25 ug/ul 12 12 37.60 0.60 12 38.970.99 12 38.24 0.50 12 40.25 0.77 1.00 ug/ul 12 12 37.17 0.60 12 38.840.69 12 38.21 0.94 12 39.62 0.85 3.00 ug/ul 12 11 38.14 1.90 12 39.030.96 12 37.83 0.68 12 40.37 1.07 5.00 ug/ul 12 12 37.19 0.60 12 38.710.73 12 38.00 1.07 12 39.67 0.82 30% 0.01 ug/ul 12 3 43.33 1.34 11 43.090.91 11 41.13 1.71 5 46.70 1.59 Wash 0.05 ug/ul 12 7 42.77 3.09 12 42.700.90 12 40.85 1.31 9 46.57 4.30 Buffer 0.10 ug/ul 12 11 40.84 1.21 1242.38 0.67 12 40.39 0.43 12 44.95 1.82 0.25 ug/ul 12 12 39.81 0.69 1242.33 0.92 12 40.14 0.69 12 42.98 1.31 1.00 ug/ul 12 12 38.20 0.64 1240.18 0.54 12 39.23 0.34 12 41.17 0.93 3.00 ug/ul 12 12 37.84 0.61 1239.69 0.57 12 38.59 0.52 12 41.78 0.96 5.00 ug/ul 12 12 37.43 0.50 1239.43 0.40 12 38.54 0.56 12 41.31 0.95

The table shows higher concentrations of cyclodextrin up to 5.0 ug/mlare effective in neutralizing SDS.

Thus, formulations such as those in Table 13 can be supplemented withcyclodextrin, e.g., at 0.1 to 5 ug/ul, or 0.1 to 0.5 cyclodextrin.

From the foregoing, it will be appreciated that, although specificembodiments have been described herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of this explicit disclosure. Accordingly, the invention is notlimited by the explicit disclosure. Unless otherwise apparent from thecontext any embodiment, feature, aspect or step can be used incombination with any other. Unless otherwise apparent from the context,any composition said to comprise enumerated components may also consistor consist essentially of those components All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entireties for all purposes.

1-163. (canceled)
 164. A composition of an aqueous solution comprising aflap endonuclease, a bulking agent, and an organic buffer, wherein theaqueous solution has an inorganic salt concentration of 5 mM or less andwherein the composition is substantially free of glycerol.
 165. Thecomposition of claim 164 wherein the flap endonuclease is a Cleavase.166. The composition of claim 165, wherein the aqueous solution furthercomprises one or more of: at least one detection probe, at least oneprobe oligonucleotide, at least one invader probe, at least onesignalling probe at least one signalling probe containing a flap region,at least one FRET probe, and at least one target capture probe.
 167. Thecomposition of claim 166, wherein the aqueous solution comprises atleast one signal probe and at least one invader probe, wherein the atleast one signal probe and the at least one invader probe are capable ofannealing to a target nucleic acid to form a three-dimensional structurethat can be recognized by the flap endonuclease.
 168. The composition ofclaim 164, further comprising at least one polymerase selected from thegroup consisting of: a hot-start polymerase, Taq DNA polymerase, Taqpolymerase wherein the polymerase is bound by an antibody thatspecifically blocks polymerase activity of the polymerase, a chemicallymodified recombinant Taq DNA polymerase wherein the chemicalmodification inhibits polymerase activity of the polymerase, a reversetranscriptase, an AMV reverse transcriptase, and an MMLP reversetranscriptase; wherein the polymerase is present at a concentration ofabout 0.1 U/μl to about 4.0 U/μl, about 0.10 U/μl to about 0.2 U/μl,about 0.14 U/μl, about 0.146 U/μl, or about 0.1686 U/μl.
 169. Thecomposition of claim 164, wherein the bulking agent is trehalose,wherein the trehalose is present at a concentration of about 0.2 M toabout 0.5 M, about 0.2 M to about 0.4 M, about 100 mM to about 300 mM,about 300 mM, about 0.36 M, or about 0.47 M.
 170. The composition claim164, wherein the inorganic salt is: (a) sodium chloride, wherein thesodium chloride is present at a mass per microliter concentration ofless than 1 mM, about 0.029 μg/μl to about 0.35 μg/μl, about 0.32 ug/ul,0.292 μg/μl or less, 0.234 μg/μl or less, 0.175 μg/μl or less, 0.146μg/μl or less, 0.117 μg/μl or less, 0.088 μg/μl or less, 0.058 μg/μl orless, 0.029 μg/μl or less, or 0.015 μg/μl or less; and/or (b) potassiumchloride, wherein the potassium chloride is present at a mass permicroliter of less than 1 mM, about 0.373 μg/μl to about 0.019 μg/μl,0.373 μg/μl or less, 0.298 μg/μl or less, 0.224 μg/μl or less, 0.186μg/μl or less, 0.149 μg/μl or less, 0.112 μg/μl or less, 0.075 μg/μl orless, 0.037 μg/μl or less, or 0.019 μg/μl or less.
 171. The compositionof claim 170, wherein the aqueous solution does not contain sodiumchloride.
 172. The composition of claim 164, wherein the aqueoussolution contains less than 1 mM magnesium ions or less than 0.1 mMmagnesium ions.
 173. The composition of claim 164, wherein the aqueoussolution further comprises one or more of dATP, dGTP, dCTP, dTTP, anddTUP; wherein the dATP, dGTP, and/or dCTP, if present, are each at aconcentration of about 0.1 mM to about 0.4 mM, about 0.29 to about 0.46mM, about 0.3 mM to about 0.4 mM, or about 0.375 mM; the dTTP, ifpresent, is at a concentration of about 0.1 mM to about 0.4 mM, about0.2 to about 0.37 mM, about 0.3 mM to about 0.4 mM, or about 0.284 mM;and the dUTP, if present, is at a concentration of about 0.1 mM to about0.4 mM, about 0.125 to about 0.234 mM, about 0.3 mM to about 04 mM, orabout 0.182 mM.
 174. The composition of claim 1, wherein the flapendonuclease is present in the aqueous solution at about 0.020 μg/μl toabout 0.040 μg/μl, about 0.030 μg/μl to about 0.04 μg/μl, or about 0.030μg/μl to about 0.035 μg/μl.
 175. The composition of claim 164, whereinthe organic buffer is selected from the group consisting of:3-(N-morpholino)propanesulfonic acid (MOPS) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, citratebuffer, acetate buffer, CHES buffer, Good's buffer, HEPES buffer, MESbuffer, tricine buffer, glycinamide buffer, succinate buffer, gluconatebuffer, and combinations thereof.
 176. The composition of claim 175,wherein the organic buffer is MOPS buffer and present at a concentrationof 10-20 mM, 12.5-20 mM, 12.5-15 mM, 15 mM, or 12.5 mM; or Tris bufferand present at a concentration of 5-60 mM, 10-30 mM, 40-60 mM, 50 mM, 20mM, or 10 mM.
 177. The composition of claim 164, wherein the compositioncontains a globular protein.
 178. The composition of claim 177, whereinthe globular protein is selected from the group consisting of: bovineserum albumin (BSA), non-acetylated BSA, and ultrapure non-acetylatedBSA
 179. The composition of claim 164, wherein the flap endonuclease isa Cleavase® enzyme, the bulking agent is trehalose, and the organicbuffer is MOPS buffer, wherein the Cleavase® is present at about0.030-0.035 μg/μl, the trehalose is present at about 0.3-0.36 M, theMOPS buffer is present at about 12.5-15 mM, and wherein the compositionfurther comprises dNTPs at a concentration of about 0.3-0.38 mM each andbovine serum albumin at a concentration of about 0.5 μg/μl
 180. Thecomposition of claim 164, wherein the aqueous solution further comprisesalpha cyclodextrin.
 181. A dried form of the composition of claim 164.182. A method for preparing a dried composition for use in performing anucleic acid based assay, the method comprising the steps of: (i)freezing an aqueous solution of claim 164, thereby forming a frozen formof the aqueous solution; and (ii) exposing the frozen form from step (i)to lyophilization conditions, thereby forming a dried composition. 183.Use of the dried composition according to claim 182, wherein the driedcomposition is combined with a reconstitution solution for performing anucleic acid based assay.